Apoptotic cell mimetics

Abstract

Provided herein are hydrogel beads that mimic living, dead and apoptotic cells. The present disclosure also provides kits and compositions of the hydrogel beads. The disclosure further includes methods of using the kit, the composition, and the hydrogel bead to determine whether a target cell sample contains one or more living cells, dead cells, or apoptotic cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 348,414, filed on June 2, 2022. The above application is incorporated herein by reference in its entirety for all purposes. Reference to an electronic sequence listing

[0002] The contents of the electronic sequence listing (SLIN_012_01WO_SeqList_ST26.xml; size: 124,536 bytes; creation date: June 2, 2023) are incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to hydrogel beads that simulate live cells, dead cells, and apoptotic cells. The present disclosure also provides kits and compositions of hydrogel beads. The present disclosure further includes methods of using the kits, compositions, and hydrogel beads to determine whether a target cell sample contains one or more live cells, dead cells, or apoptotic cells. Background Art

[0004] Flow cytometry is used to analyze and detect the chemical and physical characteristics of cells. Data from this technology allows doctors to diagnose and stage a variety of diseases, including cancer. Removing dead and dying cells from flow cytometry data is critical to ensuring the accuracy of the analysis. Dead cells are autofluorescent and difficult to eliminate from the analysis based only on forward and side scatter. In flow cytometry viability assays, cells are stained with viability dyes to identify dead and dying cells and evaluated on a flow cytometer. However, the accuracy of these viability assays requires appropriate controls. Typically, these controls require the use of purified cells of the target cell type. These purified cells are obtained using thermal or chemical methods, which are wasteful, time-consuming, not fully standardized, and prone to batch-to-batch variation. In addition, cells used for calibration may be rare or in short supply. Therefore, there is a need in the art for synthetic compositions that can be used as controls for dead cells, live cells, and dying cells. Summary of the invention

[0005] Provided herein is a hydrogel bead comprising: a) a polymerized monomer and a bifunctional monomer; and b) a pro-apoptotic signal binding agent.

[0006] Provided herein is a hydrogel bead comprising: a) a polymerized monomer and a bifunctional monomer; and b) a pro-apoptotic signal.

[0007] Provided herein is a hydrogel bead comprising: a) a polymerized monomer and a bifunctional monomer; b) a pro-apoptotic signal binding agent; and c) an encapsulated nucleic acid.

[0008] Provided herein is a hydrogel bead comprising: a) a polymerized monomer and a bifunctional monomer; b) a pro-apoptotic signal; and c) an encapsulated nucleic acid.

[0009] Provided herein is a kit comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal binding agent of the first population of hydrogel beads; and iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0010] Provided herein is a composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal binding agent of the first population of hydrogel beads; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0011] Provided herein is a kit comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal of the first population of hydrogel beads; and iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0012] Provided herein is a composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal of the first population of hydrogel beads; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0013] Provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population described herein or from a kit or composition described herein; b) contacting the hydrogel bead population with a pre-apoptotic signal and / or a DNA dye; c) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the hydrogel bead population; d) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the target cell sample; and e) comparing the concentrations of the pre-apoptotic signal and / or the DNA dye measured in the hydrogel bead population and the target cell sample; thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0014] Provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population disclosed herein or from a kit or composition disclosed herein; b) contacting the hydrogel bead population with a pre-apoptotic signal and / or a DNA dye; c) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the hydrogel bead population in a cell counting device; d) calibrating the cell counting device based on the measured concentration of the pre-apoptotic signal and / or the DNA dye of the hydrogel beads; e) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0015] Provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population disclosed herein or from a kit or composition disclosed herein; wherein at least a hydrogel bead population within the hydrogel bead population contains a pre-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the hydrogel bead population; c) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the target cell sample; and d) comparing the concentrations of the pre-apoptotic signal and / or the DNA dye measured in the hydrogel bead population and the target cell sample; thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0016] Provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population disclosed herein or from a kit or composition disclosed herein; wherein at least a hydrogel bead population within the hydrogel bead population contains a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pro-apoptotic signal and / or the DNA dye in the hydrogel bead population in a cell counting device; c) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or the DNA dye of the hydrogel beads; d) measuring the concentration of the pro-apoptotic signal and / or the DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pre-apoptotic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is shown that the hydrogel beads described herein can be tuned to match the forward scatter and side scatter of target cells such as granulocytes, monocytes, and lymphocytes.

[0018] Figure 2 Shown are hydrogel beads containing: (i) polymerized monomers and bifunctional monomers, (ii) amine dye binders, and (iii) encapsulated nucleic acids in combination with the DNA intercalating dye propidium iodide ("PI").

[0019] Figure 3 shows the PI vs. Figure 2 The mean fluorescence intensity of hydrogel-bead binding.

[0020] Figure 4 Shown are hydrogel beads containing: (i) polymerized monomers and bifunctional monomers, (ii) an amine dye binder, and (iii) encapsulated nucleic acids in combination with the DNA intercalating dye 7-aminoactinomycin D ("7AAD").

[0021] Figure 5 shows that 7AAD and Figure 4 The mean fluorescence intensity of hydrogel-bead binding.

[0022] Figure 6 Shown is a hydrogel bead containing the following and the amine-reactive vital dye GHOST DYE TM Combines: (i) polymerized monomers and bifunctional monomers, (ii) amine dye binders, and (iii) encapsulated nucleic acids.

[0023] Figure 7 Shows GHOST DYE TM and Figure 6 The mean fluorescence intensity of hydrogel-bead binding.

[0024] Figure 8Shown is a hydrogel bead containing the following with the amine-reactive vitality dye LIVE / DEAD TM Blue binding can be fixed to: (i) polymerized monomers and bifunctional monomers, (ii) amine dye binders, and (iii) encapsulated nucleic acids.

[0025] Fig. 9 Shows LIVE / DEAD TM Can be fixed with blue and Figure 8 The mean fluorescence intensity of hydrogel-bead binding.

[0026] Fig.10 Shown is a hydrogel bead containing the following and the amine-reactive vital dye GHOST DYE TM and the DNA intercalating dye 7-aminoactinomycin D ("7AAD") are combined with: (i) polymerized monomers and bifunctional monomers, (ii) amine dye binders, and (iii) encapsulated nucleic acids.

[0027] Fig.11 The DNA intercalating dye 7AAD is shown Fig.10 The mean fluorescence intensity of hydrogel-bead binding.

[0028] Fig.12 Shown is the amine-reactive vital dye GHOST DYE TM and Fig.10 The mean fluorescence intensity of hydrogel-bead binding.

[0029] Fig.13 Forward scatter and side scatter of lymphocytes are shown.

[0030] Fig.14 Forward scatter and side scatter of the hydrogel beads of Example 4 are shown.

[0031] Fig.15 Forward and side scatter are shown for amine-reactive polystyrene beads.

[0032] Fig.16 The mean fluorescence intensity of the DNA intercalating dye 7AAD binding to the mixture of live lymphocytes and heat-killed lymphocytes of Example 4 is shown.

[0033] Fig.17 The average fluorescence intensity of the DNA intercalating dye 7AAD bound to the hydrogel beads of Example 4 is shown.

[0034] Fig.18 Shown is the amine-reactive vital dye GHOST DYE TM The mean fluorescence intensity of the mixture of live lymphocytes and heat-killed lymphocytes of Example 4.

[0035] Fig.19Shown is the amine-reactive vital dye GHOST DYE TM Mean fluorescence intensity of binding to the hydrogel beads of Example 4.

[0036] Fig. 20 Shown is the amine-reactive vital dye GHOST DYE TM Mean fluorescence intensity of binding to the polystyrene beads of Example 4.

[0037] Fig.21 The binding of a hydrogel bead composition containing three hydrogel bead populations to annexin V is shown, and the annexin V is labeled with fluorescein isothiocyanate ("FITC") dye (y-axis) and DNA intercalating dye 7AAD (x-axis). The first hydrogel bead population contains both a pre-apoptotic signal binder and an encapsulated nucleic acid. The second nucleic acid bead population contains a pre-apoptotic signal binder, but does not contain encapsulated amino acids. The third hydrogel bead population contains neither a pre-apoptotic signal binder (e.g., phosphatidylserine ("PS")) nor encapsulated nucleic acids. The first hydrogel bead population binds to both annexin V and 7AAD, and acts as a dead cell mimic. The second hydrogel bead population binds to annexin V but does not bind to 7AAD, and acts as an apoptotic cell mimic. The third hydrogel bead population is neither bound to annexin V nor to 7AAD, and acts as a living cell mimic.

[0038] Figure 22-Figure 26 Shown on day 0 ( Fig. 22 ) and one day after storage ( Fig.23 ), seven days( Fig.24 ), Fourteen days( Fig.25 ) and thirty-seven days ( Fig.26 ), the hydrogel bead composition binds to annexin V and 7AAD. The first hydrogel bead population contains both a pro-apoptotic signal binding agent (e.g., an anti-annexin V antibody or an antigen-binding fragment thereof) and an encapsulated nucleic acid. The second hydrogel bead population contains a pro-apoptotic signal binding agent (e.g., an anti-annexin V antibody or an antigen-binding fragment thereof) but does not contain an encapsulated nucleic acid. The third hydrogel bead population contains neither a pro-apoptotic signal binding agent (e.g., phosphatidylserine ("PS")) nor an encapsulated nucleic acid.

[0039] Figure 27-Figure 29 Shown in BD FACSLyric TM Binding of 7AAD on the cell counting device to the following samples stained with 7AAD alone: ​​Hydrogel bead composition ( Fig. 27 ), Jurkat cells ( Fig.28 ) and PBMC( Fig.29). The hydrogel bead composition contains three hydrogel bead populations. The first hydrogel bead population contains both a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) and an encapsulated nucleic acid. The second hydrogel bead population contains a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof), but does not contain an encapsulated nucleic acid. The third hydrogel bead population contains neither a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) nor an encapsulated nucleic acid.

[0040] Figure 30-Figure 31 Shown in Binding of 7AAD on the Aurora Cytometry Device to the following samples stained with 7AAD alone: ​​Hydrogel Bead Composition ( Fig.30 ) and Jurkat cells ( Fig.31 ). The hydrogel bead composition contains three hydrogel bead populations. The first hydrogel bead population contains both a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) and an encapsulated nucleic acid. The second hydrogel bead population contains a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof), but does not contain an encapsulated nucleic acid. The third hydrogel bead population contains neither a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) nor an encapsulated nucleic acid.

[0041] Figure 32-Figure 34 Shown in BD FACSLyric TM Binding of Annexin V to the following composition on a cell counting device: a composition containing three hydrogel bead populations ( Fig.32 ), Jurkat cells ( Fig.33 ) or PBMC( Fig.34 ). The first hydrogel bead population contains both a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) and an encapsulated nucleic acid. The second hydrogel bead population contains a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof), but does not contain an encapsulated nucleic acid. The third hydrogel bead population contains neither a pro-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) nor an encapsulated nucleic acid.

[0042] Figure 35-Figure 36 Shown in Annexin V and a composition containing three hydrogel bead populations on the Aurora cell counting device ( Fig.35 ) and Jurkat cells ( Fig.36). The first hydrogel bead population contains both a pro-apoptotic signal binding agent (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) and an encapsulated nucleic acid. The second hydrogel bead population contains a pro-apoptotic signal binding agent (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) but does not contain an encapsulated nucleic acid. The third hydrogel bead population contains neither a pro-apoptotic signal binding agent (e.g., phosphatidylserine or an anti-annexin V antibody or an antigen-binding fragment thereof) nor an encapsulated nucleic acid.

[0043] Figure 37-Figure 39 As shown in the BD FACSLyric TM The following forward scatter and side scatter were measured on a cell counting device: Compositions containing three hydrogel bead populations ( Fig.37 ); Jurkat cells ( Fig.38 ); or peripheral blood mononuclear cells (PBMC) ( Fig.39 Each composition was gated to exclude debris (see Figure 37-Figure 39 Each of these compositions is described in detail in Example 8.

[0044] Figure 40-Figure 42 As shown in the BD FACSLyric TM The following forward scatter and side scatter were measured on a cell counting device: Compositions containing three hydrogel bead populations ( Fig.40 ); Jurkat cells ( Fig.41 ); or peripheral blood mononuclear cells (PBMC) ( Fig.42 Each composition was gated to exclude debris (see Figure 40-Figure 42 Each of these compositions is described in detail in Example 8.

[0045] Figure 43-Figure 45 is a histogram showing the TM The mean fluorescence intensity of 7AAD in the following compositions containing three hydrogel bead populations ( Fig.43 ); Jurkat cells ( Fig.44 ); or peripheral blood mononuclear cells (PBMC) ( Fig.45 ). Each of these compositions is described in detail in Example 8.

[0047] Figure 46-Figure 48 is a histogram that shows that The mean fluorescence intensity of 7AAD in the following compositions containing three hydrogel bead populations ( Fig.46 ); Jurkat cells ( Fig.47 ); or peripheral blood mononuclear cells (PBMC) ( Fig.48 ). Use a single dye (7AAD or The fluorescence spectrum information of Jurkat cells labeled with 647 dye-labeled annexin V) was spectrally unmixed. Each of these compositions is described in detail in Example 8.

[0048] Figure 49-Figure 51 is a histogram that shows that The mean fluorescence intensity of 7AAD in the following compositions containing three hydrogel bead populations ( Fig.49 ); Jurkat cells ( Fig.50 ); or peripheral blood mononuclear cells (PBMC) ( Fig.51 ). Use a single dye (7AAD or ALEXA The fluorescence spectrum information of PBMCs labeled with 647 dye-labeled annexin V was spectrally unmixed. Each of these compositions is described in detail in Example 8.

[0049] Figure 52-Figure 54 is a histogram showing the TM The following measurements were made using ALEXA on the cell counting device: Mean fluorescence intensity of 647 dye-labeled annexin V: Compositions containing three hydrogel bead populations ( Fig.52 ); Jurkat cells ( Fig.53 ); or peripheral blood mononuclear cells (PBMC) ( Fig.54 ). Each of these compositions is described in detail in Example 8.

[0050] Figure 55-Figure 57 is a histogram that shows that The following measurements were made using the Aurora cell counting device: Mean fluorescence intensity of 647 dye-labeled annexin V: Compositions containing three hydrogel bead populations ( Fig.55 ); Jurkat cells ( Fig.56 ); or peripheral blood mononuclear cells (PBMC) ( Fig.57 ). Use the ALEXA The fluorescence spectrum information of Jurkat cells labeled with 647 dye annexin V was spectrally unmixed. Each of these compositions is described in detail in Example 8.

[0051] Figure 58-Figure 60 is a histogram that shows that The following measurements were made using the Aurora cell counting device: Mean fluorescence intensity of 647 dye-labeled annexin V: Compositions containing three hydrogel bead populations ( Fig.58 ); Jurkat cells ( Fig.59 ); or peripheral blood mononuclear cells (PBMC) ( Fig.60 ). Use the ALEXA The fluorescence spectrum information of PBMCs labeled with 647 dye Annexin V was spectrally unmixed. Each of these compositions is described in detail in Example 8.

[0052] Figure 61-Figure 63 As shown in the BD FACSLyric TM 7AAD measured on a cell counting device and with ALEXA 647 dye-labeled annexin V binding to the following: Composition containing three hydrogel bead populations ( Fig.61 ); Jurkat cells ( Fig.62 ); or peripheral blood mononuclear cells (PBMC) ( Fig.63 ). Live cells, dead cells, and apoptotic cells (labeled as "pre-apoptotic") are labeled in each figure. BD FACSLyric TM Fluorescence spillover on a cell counting device. Each of these compositions is described in detail in Example 8.

[0053] Figure 64-Figure 66 As shown in 7AAD measured on the Aurora cell counting device and with ALEXA 647 dye-labeled annexin V binding to the following: Composition containing three hydrogel bead populations ( Fig.64 ); Jurkat cells ( Fig.65 ); or peripheral blood mononuclear cells (PBMC) ( Fig.66 ). Each of these compositions is described in detail in Example 8. In each figure, live cells, dead cells, and apoptotic cells (labeled "pre-apoptotic") are labeled. Fluorescence spectra of Jurkat cells labeled with 647 dye Annexin V and / or 7AAD were spectrally unmixed.

[0054] Figure 67-Figure 69 As shown in 7AAD measured on the Aurora cell counting device and with ALEXA 647 dye-labeled annexin V binding to the following: Composition containing three hydrogel bead populations ( Fig.67); Jurkat cells ( Fig.68 ); or peripheral blood mononuclear cells (PBMC) ( Fig.69 ). Each of these compositions is described in detail in Example 8. In each figure, live cells, dead cells, and apoptotic cells (labeled "pre-apoptotic") are labeled. Fluorescence spectra of PBMCs labeled with 647 dye Annexin V and / or 7AAD were spectrally unmixed.

[0055] Figure 70-Figure 72 As shown in 7AAD measured on the Aurora cell counting device and with ALEXA 647 dye-labeled annexin V binding to the following: Composition containing three hydrogel bead populations ( Fig.70 ); Jurkat cells ( Fig.71 ); or peripheral blood mononuclear cells (PBMC) ( Fig.72 ). Each of these compositions is described in detail in Example 8. In each figure, live cells, dead cells, and apoptotic cells (labeled "pre-apoptotic") are labeled. Fluorescence spectra of 647 dye-labeled Annexin V and / or 7AAD containing compositions of three hydrogel bead populations were spectrally unmixed.

[0056] Fig.73 Forward and side scatter of five-day-old Jurkat cells are shown.

[0057] Figure 74-Figure 76 Tuning of the optical properties of hydrogel beads to match those of target cells is shown. DETAILED DESCRIPTION

[0058] The present disclosure provides hydrogel beads (Part IV) and kits (Part VI) and compositions (Part VII) comprising the hydrogel beads. The present disclosure also provides methods of using the hydrogel beads and kits and compositions (Part VIII) comprising the hydrogel beads.

[0059] The hydrogel beads provided herein simulate living cells, dead cells or apoptotic cells. The optical scattering properties (e.g., forward scattering and / or side scattering) of these hydrogel beads can be tuned to match those optical scattering properties of the target cell population. The properties of these beads are further described in Part V.

[0060] Advantageously, these hydrogel beads and compositions and kits comprising the hydrogel beads can be used to determine whether the target cell population includes live cells, dead cells and / or cells that are undergoing apoptosis but not yet dead. Additionally, hydrogel beads and compositions and kits comprising the hydrogel beads can be used to quantify the number of live cells, dead cells and / or apoptotic cells in the target cell population. Due to several reasons, the composition comprising hydrogel beads is superior to the composition comprising cells. First, the number of hydrogel beads acting as live cell controls, dead cell controls and apoptotic cell controls can be adjusted. In contrast, the amount of dead cells, live cells and apoptotic cells acting as apoptotic controls in the cell population cannot be accurately controlled. Therefore, a hydrogel bead composition can be produced, wherein the beads acting as dead cells, live cells and apoptotic cell mimics each account for 33%. Secondly, the composition comprising hydrogel beads stained with pre-apoptotic signals and viability dyes shows clear positive and negative bead populations. In contrast, the separation between positive and negative cell populations is not very clear. Third, the composition comprising hydrogel beads shows a smaller variability than cells. Depending on the age of the cells, different batches of cells can show different characteristics, and the hydrogel bead composition can be stable for at least 37 days. Fourth, using a hydrogel bead composition as an apoptosis control is less time-consuming than using a cell population as an apoptosis control because the hydrogel bead composition does not require cell culture or induction of apoptosis. In contrast, when using a cell population as an apoptosis control, apoptosis must be induced in the control cells using thermal or chemical methods. This process is time-consuming, wasteful, and not adequately standardized. I. Definitions

[0061] The indefinite articles “a” and “an” and the definite article “the” are intended to include both the singular and the plural, unless the context in which they are used clearly indicates otherwise.

[0062] “At least one” and “one or more” are used interchangeably to mean that an item may contain one or more than one of the listed elements.

[0063] As used herein, the term "about" means plus or minus 10% of the referenced number, unless otherwise stated or otherwise apparent from the context, and except where such range would exceed 100% of a possible value or be below 0% of a possible value, such as less than 0% content of an ingredient, or greater than 100% of the total content of the composition. For example, reference to about 10% monomer by weight of the hydrogel means that the monomer may be present in any amount ranging from 9% to 11% by weight of the hydrogel. Unless otherwise indicated, it should be understood that all numbers used in the specification and claims expressing quantities, ratios and numerical properties of ingredients, reaction conditions, and the like are intended to be modified in all instances by the term "about".

[0064] The term "scattering adjustment additive" refers to any element capable of adjusting the lateral scattering of the hydrogel beads. Non-limiting examples of scattering adjustment additives include nanoparticles such as those made of polymethyl methacrylate (PMMA), polystyrene (PS) or silica; and / or high refractive index molecules added to the hydrogel such as alkyl acrylates, alkyl methacrylates, vinyl aromatics (such as styrene and methyl styrene, optionally substituted on the aromatic ring by alkyl groups (such as methyl, ethyl or tert-butyl) or by halogens (such as chlorostyrene)).

[0065] The term "hydrogel beads" refers to particles made of hydrogel material and optionally containing one or more additional elements for use in the described cytometry or coulter assays. In some embodiments, the hydrogels of the present disclosure are generally spherical in shape and may resemble one or more target cells.

[0066] The term "optical scattering properties" refers to the forward scatter (FSC) and side scatter (SSC) properties of cells or hydrogels.

[0067] The term "dead cell" refers to a non-viable cell. In some embodiments, a dead cell has a permeable, disrupted, or absent membrane, such that the cytoplasm and nucleus are accessible to one or more viability dyes / markers.

[0068] The term "pre-apoptotic cell" refers to a cell that has been triggered to undergo apoptosis but has not yet died.

[0069] The term "hydrogel" refers to a material comprising a three-dimensional network of macromolecules that allows it to swell in the presence of water (i.e., a "hydrated state"), shrink in the absence of water (or by reducing the amount of water) (i.e., a "dehydrated state"), but is insoluble in water. As used herein, the term "hydrogel" refers to a material in its hydrated or dehydrated state. Swelling or water absorption is the result of the presence of hydrophilic functional groups attached to or dispersed in the macromolecular network. Crosslinking between adjacent macromolecules causes the water insolubility of these hydrogels. Crosslinking can be due to chemical bonds (i.e., covalent bonds) or physical bonds (i.e., van der Waals forces, hydrogen bonding, ionic forces, etc.). These chemical crosslinks can also be hydrolyzed under certain conditions, thereby reversing the insolubility of the hydrogel. A variety of chemical crosslinking chemistries are described in the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bondingcrosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf), the disclosure of which is incorporated by reference in its entirety for all purposes.

[0070] The term "bifunctional monomer" refers to a monomer containing a first functional group and a second functional group, wherein the first functional group is polymerized with the monomer to form a hydrogel. In embodiments, the second functional group can be used to conjugate a fluorophore or a cell surface receptor or a domain thereof.

[0071] The term "forward scatter" refers to the light scattering properties of a material measured in the direction parallel to the propagation of the light. Forward scatter is a common measure of particle size and may also be affected by the refractive index of the hydrogels of the present disclosure.

[0072] The term "side scatter" refers to the light scattering properties of a material measured in the perpendicular direction of light propagation. Side scattering is a common measure of particle complexity and may also be affected by the refractive index of the hydrogels of the present disclosure.

[0073] The term "substantially similar" means at least 40% similar, at least 50% similar, at least 60% similar, at least 70% similar, at least 80% similar, at least 90% similar, at least 95% similar, at least 96% similar, at least 97% similar, at least 98% similar, or at least 99% similar.

[0074] The term "cell counting device" refers to a device for measuring the number and characteristics of cells. Variables that can be measured by cell counting methods include cell size, cell count, cell morphology (shape and structure), cell cycle stage, DNA content, and the presence or absence of specific proteins on the cell surface or in the cytoplasm. A common cell counting device according to the present disclosure is a flow cytometer. Flow cytometers are well known in the art and typically include a light source, optics, and a stream flow.

[0075] The term "antigen-binding fragment" refers to a polypeptide fragment containing at least one complementarity determining region (CDR) of an immunoglobulin heavy chain and / or light chain that binds to at least one epitope of an antigen of interest. In this regard, an antigen-binding fragment of an anti-Annexin V antibody may comprise 1, 2, 3, 4, 5, or all 6 CDRs from variable heavy chain (VH) and variable light chain (VL) sequences of an antibody that specifically binds to Annexin V. Antigen-binding fragments include proteins that comprise a portion of a full-length antibody (usually its antigen-binding or variable region), such as Fab, F(ab')2, Fab', Fv fragments, miniantibodies, diabodies, single domain antibodies (dAbs), single chain variable fragments (scFvs), and multispecific antibodies formed from antibody fragments.

[0076] In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to the percentage of two or more sequences or subsequences that have a specified number of identical nucleotide or amino acid residues when compared. Unless otherwise indicated, percent identity is calculated using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool. (available at blast.ncbi.nlm.nih.gov / Blast.cgi, version BLAST+2.13.0). II. Apoptosis

[0077] Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and cell death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA degradation. The average adult loses billions of cells every day due to apoptosis.

[0078] Compared with necrosis (which is a form of traumatic cell death caused by acute cell injury), apoptosis is a highly regulated and controlled process that has advantages during the life cycle of an organism. For example, the separation of fingers and toes in a developing human embryo occurs because the cells between the toes undergo apoptosis. Unlike necrosis, apoptosis produces cell fragments called apoptotic bodies, which phagocytes are able to engulf and remove before the cell contents can overflow into surrounding cells and cause damage to surrounding cells.

[0079] Apoptosis is a highly regulated process. Apoptosis can be triggered by one of two pathways: intrinsic and extrinsic. In the intrinsic pathway, cells kill themselves because they feel cell stress, while in the extrinsic pathway, cells kill themselves because of signals from other cells. Weak external signals may also activate the intrinsic pathway of apoptosis. Both pathways induce cell death by activating caspases (which are proteases) or enzymes that degrade proteins. Both pathways activate initiating caspases, which then activate execution caspases, which then kill cells by indiscriminately degrading proteins. The membrane of the cell is subjected to such degradation, resulting in the release of nucleic acids (such as nuclear DNA, mitochondrial DNA, and RNA).

[0080] The intrinsic pathway is also called the mitochondrial pathway. Mitochondria are essential for multicellular life. Without mitochondria, cells cease aerobic respiration and die rapidly. This fact forms the basis of some apoptotic pathways. Apoptotic proteins targeted to mitochondria affect mitochondria in different ways. The apoptotic proteins can cause mitochondrial swelling through the formation of membrane pores, or the apoptotic proteins can increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.

[0081] During apoptosis, cytochrome c is released from mitochondria by the action of the proteins Bax and Bak. The mechanism of this release is mysterious, but appears to stem from the large number of Bax / Bak homodimers and heterodimers that insert into the outer membrane. Once cytochrome c is released, it binds to apoptotic protease activating factor-1 (Apaf-1) and ATP, and then to pro-caspase-9 to produce a protein complex called the apoptosome. The apoptosome cleaves pro-caspase into its active form, caspase-9, which in turn cleaves pro-caspase and activates it into effector caspase-3.

[0082] As the permeability of the mitochondrial membrane increases, the mitochondria also release a protein called SMAC (second mitochondrial-derived caspase activator) into the cytosol of the cell. SMAC binds to proteins that inhibit apoptosis (IAPs), thereby inactivating them and preventing IAPs from blocking the process and thus allowing apoptosis to proceed. IAPs also typically inhibit the activity of a group of cysteine ​​proteases called caspases (which perform cell degradation). Therefore, it can be seen that the actual degradation enzymes are indirectly regulated by mitochondrial permeability.

[0083] Two theories for the direct extrinsic initiation of apoptotic mechanisms in mammals have been proposed: the TNF (tumor necrosis factor)-induced model and the Fas-Fas ligand-mediated model, both involving receptors of the TNF receptor (TNFR) family coupled to extrinsic signals.

[0084] TNF-α is a cytokine produced primarily by activated macrophages and is the main extrinsic mediator of apoptosis. Most cells in the human body have two receptors for TNF-α: TNFR1 and TNFR2. It has been shown that the binding of TNF-α to TNFR1 triggers a pathway leading to caspase activation via the intermediate membrane proteins TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). cIAP1 / 2 can inhibit TNF-α signaling by binding to TRAF2. FLIP inhibits the activation of caspase-8. Binding of this receptor can also indirectly lead to the activation of transcription factors involved in cell survival and inflammatory responses. However, signaling via TNFR1 may also induce apoptosis in a caspase-independent manner.

[0085] The fas receptor (first apoptotic signal) (also known as Apo-1 or CD95) is a transmembrane protein of the TNF family that binds to the Fas ligand (FasL). The interaction between Fas and FasL leads to the formation of the death-inducing signaling complex (DISC), which contains FADD, caspase-8, and caspase-10. In some types of cells (type I), processed caspase-8 directly activates other members of the caspase family and triggers the execution of apoptosis of the cells. In other types of cells (type II), Fas-DISC initiates a feedback loop that gradually leads to the increased release of pro-apoptotic factors from mitochondria and the amplified activation of caspase-8.

[0086] Defective apoptosis is involved in a variety of diseases. Excessive apoptosis leads to atrophy, while insufficient apoptosis can lead to uncontrolled cell proliferation, such as cancer. The development of AIDS from human immunodeficiency virus infection is mainly due to the rapid depletion of CD4+ T helper lymphocytes, which prevents the body's bone marrow from replenishing the cells, resulting in a damaged immune system. One of the mechanisms for depleting T helper cells is apoptosis, which is caused by a series of biochemical pathways.

[0087] Inhibition of apoptosis can lead to many cancers, inflammatory diseases and viral infections. Interruption of the process may cause the lifespan of cells to exceed their "use-by date" and be able to replicate and pass on any defective mechanisms to their offspring, thereby increasing the likelihood that the cells will become cancerous or diseased. The associated accumulation of cells was initially thought to be due to an increase in cell proliferation, but it is now believed that this is also due to a decrease in cell death. The most common of these diseases is cancer, which is a disease of excessive cell proliferation, which is often characterized by overexpression of members of the IAP family. Therefore, malignant cells undergo an abnormal response to apoptosis induction: cycle regulatory genes (such as p53, ras or c-myc) are mutated or inactivated in diseased cells, and other genes (such as bcl-2) also change their expression in tumors. Some apoptotic factors are essential during mitochondrial respiration, such as cytochrome C. Pathological inactivation of apoptosis in cancer cells is associated with a frequent shift of respiratory metabolism to glycolysis (an observation known as the "Warburg hypothesis").

[0088] Some factors such as Fas receptor and caspase promote apoptosis, while some members of the Bcl-2 protein family inhibit apoptosis. Phosphatidylserine (PS) is a common marker of apoptosis. In normal healthy cells, PS is present in the intracellular side of the cell membrane. However, during apoptosis, PS translocates to the extracellular side of the membrane. The connection between TNF-α and apoptosis shows why the abnormal production of TNF-α plays a fundamental role in several human diseases, especially in autoimmune diseases. Known apoptosis is one of the main mechanisms of targeted cancer therapy. Recently designed luminescent iridium complex-peptide hybrid (IPH), it simulates TRAIL and binds to the death receptor on cancer cells, thereby inducing apoptosis of cancer cells.

[0089] The hydrogel beads and compositions and kits comprising the hydrogel beads can be used to detect apoptosis, dead cells and live cells. III. Detection of apoptosis using viability staining

[0090] Viability staining is the process of distinguishing live cell populations from dead cell populations within a sample. Viability controls are typically a mixture of live and dead cells stained with a differentiating dye. These dyes typically fall into one of two major categories: DNA intercalating stains and primary amine (protein) stains. Despite the fact that several DNA binding dyes are commonly used to identify live cell populations, there are very few compensation beads for DNA dyes. Furthermore, existing polystyrene beads for amine-reactive dyes have fundamentally different properties when compared to cellular material, creating difficulties when trying to work with certain major classes of dyes in critical fluorescence channels.

[0091] In order to create staining controls, apoptosis must be induced in real cells using thermal or chemical methods, which is a wasteful, time-consuming, and not fully standardized process. A mixture of live and treated cells is stained with a fluorescent annexin V conjugate, and this is used to distinguish apoptotic from non-apoptotic populations. In flow cytometry, these controls can be used to establish compensation. In addition to annexin V, stains that are impermeable to live cells (such as propidium iodide or 7AAD) are often used to eliminate false positives. In the late stages of apoptosis, the cell membrane will begin to rupture, and annexin V enters the cell and can bind to PS on the intracellular portion of the membrane. Stains that are impermeable to live cells are used to detect membrane rupture.

[0092] The two dyes are often sold together in "apoptosis detection" kits. There are existing bead products associated with Annexin V, but they are all intended for use in either depleting or selecting apoptotic cells in a population. For example, Miltenyi Biotec sells Annexin V microbeads in which Annexin V is conjugated to the surface of the beads, but the beads are used to enrich for apoptotic cell populations, not as a compensation control by themselves.

[0093] The ability to identify and measure specific cell types (such as apoptotic cells) relies on the proper calibration of the measuring instrument. Calibration relies on the use of purified cells of the cell type of interest. These purified cells are obtained using thermal or chemical methods, which are wasteful, time consuming, not fully standardized, and prone to batch-to-batch variation. In addition, the cells used for calibration may be rare or in short supply. Therefore, there is a need in the art for synthetic compositions with tunable optical and binding properties that can simulate apoptotic cells.

[0094] Flow cytometry is a technique that allows rapid separation, counting and characterization of individual cells (such as potential apoptotic cells) and is commonly used in clinical and laboratory settings for a variety of applications. Flow cytometry based on optics relies on directing a beam to a fluid stream of hydrodynamic focusing. Multiple detectors are then aimed at the point where the stream passes through the beam: one is consistent with the beam (forward scatter or FSC) and several are perpendicular to the beam (side scatter or SSC). FSC is related to cell volume, and SSC depends on the internal complexity of the particle (e.g., the shape of the nucleus, the amount and type of cytoplasmic particles or membrane roughness). Due to these correlations, different specific cell types exhibit different FSC and SSC, thereby allowing cell types to be distinguished.

[0095] Using cells of interest to create calibration controls is time consuming, wasteful, and not adequately standardized. Using polystyrene beads as calibration controls results in controls that have fundamentally different properties when compared to the cell material of interest, thus creating difficulties when trying to work with certain major classes of dyes in key fluorescence channels.

[0096] Provided herein are calibration controls for flow cytometry that avoid problems associated with using cells of interest. The calibration controls described herein include hydrogel beads. IV. Hydrogel Beads

[0097] Provided herein are hydrogel beads comprising (a) polymerized monomers and bifunctional monomers; and in some embodiments (b) a pro-apoptotic signal binder. Also provided herein are hydrogel beads comprising (a) polymerized monomers and in some embodiments bifunctional monomers; and (b) a pro-apoptotic signal. In embodiments, the hydrogel beads comprise (c) an encapsulated nucleic acid. In embodiments, the hydrogel beads comprise (d) an additional component. IV-A. Polymerized Monomers and Bifunctional Monomers

[0098] In embodiments, the polymerized monomers and bifunctional monomers of hydrogel beads described herein form hydrogels.Hydrogels are materials comprising macromolecular three-dimensional networks, which allow them to swell in the presence of water (i.e., "hydrated state"), shrink in the absence of water (or by reducing the amount of water) (i.e., "dehydrated state"), but are insoluble in water. As used herein, the term "hydrogel" refers to a material in its hydrated or dehydrated state. Swelling or water absorption is the result of the presence of hydrophilic functional groups attached to or dispersed in the macromolecular network. Crosslinking between adjacent macromolecules causes the water insolubility of these hydrogels. Crosslinking can be due to chemical bonds (i.e., covalent bonds) or physical bonds (i.e., van der Waals forces, hydrogen bonding, ionic forces, etc.). These chemical crosslinks can also be hydrolyzed under certain conditions, thereby reversing the insolubility of hydrogels. A variety of chemical crosslinking chemistries are described in the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf), the disclosure of which is incorporated by reference in its entirety for all purposes.

[0099] In embodiments, the hydrogel comprises greater than about 30%, greater than about 40%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% water by weight. In embodiments, the hydrogel has a water content of from about 10% by weight to about 95% by weight, or from about 20% by weight to about 95% by weight, or from about 30% by weight to about 95% by weight, or from about 40% by weight to about 95% by weight, or from about 50% by weight to about 95% by weight, or from about 60% by weight to about 95% by weight, or from about 70% by weight to about 95% by weight, or from about 80% by weight to about 95% by weight.

[0100] In an embodiment, the hydrogel maintains the same shape under dehydrated and hydrated conditions. For example, if the hydrogel has an approximately spherical shape under dehydrated conditions, it will be approximately spherical under hydrated conditions.

[0101] In embodiments, the hydrogel forms particles. In embodiments, the hydrogel is synthesized by polymerizing one or more monomers provided herein. In embodiments, any form of polymerization known to those skilled in the art can be used to form the polymer. In embodiments, the polymerization is catalyzed by ultraviolet light induced free radical formation and reaction progression. In embodiments, acrylates are monomers that are polymerized. In embodiments, acrylamide is a monomer that is polymerized.

[0102] In an embodiment, the acrylamide is a polymerizable carbohydrate-derived acrylamide, as described in U.S. Pat. No. 6,107,365, the disclosure of which is incorporated by reference in its entirety for all purposes. As described therein and known to those of ordinary skill in the art, the specific attachment of an acrylamide group to a sugar is readily adaptable to a range of monosaccharides and higher order polysaccharides, such as synthetic polysaccharides or polysaccharides derived from natural sources, such as glycoproteins found in serum or tissues.

[0103] In embodiments, acrylate functionalized poly(ethylene glycol) monomers are polymerized. In embodiments, acrylamide functionalized PEG is polymerized.

[0104] In embodiments, the hydrogel beads comprise a monofunctional monomer polymerized with at least one difunctional monomer. In embodiments, the hydrogel beads comprise a polymer of acrylamide (ie, poly-acrylamide) and bisacrylamide (a difunctional monomer).

[0105] In embodiments, hydrogel beads provided herein include bifunctional monomers polymerized with a second bifunctional monomer. In embodiments, hydrogel beads including bifunctional monomers polymerized with a second bifunctional monomer include polymers with a mixed composition, and the mixed composition contains compatible chemicals, such as acrylamide, bisacrylamide, and bisacrylamide structural homologues containing other chemicals of a wide range. In embodiments, one functional group of the bifunctional monomer is an alkyne, and another functional group is an ester, amide, ketone, aldehyde, azide, olefin, alcohol, amine, or carboxylic acid. In embodiments, one functional group of the bifunctional monomer is an alkyne, and another functional group is an alcohol, amine, or carboxylic acid. In embodiments, one functional group of the bifunctional monomer is an olefin, and another functional group is an amine. In embodiments, one functional group of the bifunctional monomer is an olefin, and another functional group is an alcohol, amine, or carboxylic acid. In embodiments, one functional group of the bifunctional monomer is an olefin, and another functional group is an alcohol, amine, or carboxylic acid. In embodiments, one functional group of the bifunctional monomer is an olefin, and another functional group is an ester, amide, ketone, aldehyde, azide, olefin, alcohol, amine, or carboxylic acid.

[0106] In an embodiment, the hydrogel beads provided herein comprise a polymerized monofunctional monomer and are monofunctional acrylic monomers. Non-limiting examples of monofunctional acrylic monomers for use herein are acrylamide; methacrylamide; N-alkyl acrylamides, such as N-ethyl acrylamide, N-isopropyl acrylamide or N-tert-butyl acrylamide; N-alkyl methacrylamides, such as N-ethyl methacrylamide or N-isopropyl methacrylamide; N,N-dialkyl acrylamides, such as N,N-dimethyl acrylamide and N,N-diethyl-acrylamide; N-[(dialkylamino)alkyl]acrylamide, such as N-[3-(dimethylamino)propyl]acrylamide; amine or N-[3-(diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamide, such as N-[3-(dimethylamino)propyl]methacrylamide or N-[3-(diethylamino)propyl]methacrylamide; (dialkylamino)alkyl acrylates, such as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propyl acrylate or 2-(diethylamino)ethyl acrylate; and (dialkylamino)alkyl methacrylates, such as 2-(dimethylamino)ethyl methacrylate.

[0107] In an embodiment, the difunctional monomer is selected from the group consisting of: allylamine, allyl alcohol, allyl isothiocyanate, allyl chloride, and allyl maleimide.

[0108] In an embodiment, the difunctional monomer is a difunctional acrylic monomer. Non-limiting examples of difunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-bisacrylcystamine, N,N'-propylenebisacrylamide and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0109] Higher order branched and linear comonomers in the polymer blend may be substituted to tailor the refractive index while maintaining polymer density as described in US Pat. No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0110] In one embodiment, the biomonomer is functionalized with acrylamide or acrylate. For example, in one embodiment, the polymerizable acrylamide-functionalized biomolecule is an acrylamide- or acrylate-functionalized protein (e.g., an acrylamide-functionalized collagen or a functionalized collagen domain), an acrylamide- or acrylate-functionalized peptide, or an acrylamide- or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide.

[0111] Any monosaccharide, disaccharide or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In one embodiment, a monosaccharide, disaccharide or polysaccharide functionalized with acrylamide or acrylate is used as a polymerizable hydrogel monomer. In one embodiment, a structural polysaccharide is used as a polymerizable hydrogel monomer. In a further embodiment, the structural polysaccharide is arabinoxylan, cellulose, chitin or pectin. In another embodiment, alginic acid (alginate) is used as a polymerizable hydrogel monomer. In another embodiment, glycosaminoglycan (GAG) is used as a polymerizable monomer in a hydrogel provided herein. In a further embodiment, GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate or hyaluronic acid (hyaluronic acid) (also referred to as hyaluronic acid (hyaluron) or hyaluronic acid (hyaluronate) in the art), used as a polymerizable hydrogel monomer. Other compatible biomonomers and reactive chemicals thereof are known to those skilled in the art, and follow the general chemical reactivity principle.

[0112] In one embodiment, biocompatible monomers for use with the hydrogels described herein include ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidone (N-VP), styrene, or a combination thereof.

[0113] Naturally occurring hydrogels that can be used for the present disclosure include various polysaccharides that can be obtained from natural sources (such as plants, algae, fungi, yeasts, marine invertebrates and arthropods). Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derivatized starch, etc. These will generally have repeated glucose units as the main part of the polysaccharide backbone. The cross-linking chemicals of such polysaccharides are known in the art, see, for example, Thermo Scientific Crosslinking Technical Handbook entitled "Easymolecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0114] In one embodiment, hyaluronic acid is used as a hydrogel monomer (as a single monomer or as a comonomer). In one embodiment, hyaluronic acid is functionalized, for example, functionalized by acrylate or acrylamide. Hyaluronic acid is a high molecular weight GAG, consisting of disaccharide repeating units of N-acetylglucosamine and glucuronic acid, which are linked together by alternating β-1,4 and β-1,3 glycosidic bonds. In the human body, hyaluronic acid is present in several soft connective tissues, including skin, umbilical cord, synovial fluid and vitreous fluid. Therefore, in one embodiment, in the case of one or more optical properties of skin cells, umbilical cord cells or vitreous fluid cells that are expected to be simulated, in one embodiment, hyaluronic acid is used as a hydrogel monomer. The method for making hydrogel particles is described in Xu et al. (2012). Soft Matter. 8, pp. 3280-3294, and the disclosure of the document is incorporated herein in its entirety for all purposes. As described therein, hyaluronic acid can be derived with various reaction pathways, depending on the desired cross-linking chemicals and other monomers used to form hydrogel particles.

[0115] In yet other embodiments, chitosan, a linear polysaccharide composed of randomly distributed β-(1-4) linked D-glucosamine (deacetylated units) and N-acetyl-D-glucosamine (acetylated units), is used as a hydrogel monomer (either as a single monomer or as a comonomer).

[0116] Other polysaccharides for use as monomers or co-monomers in hydrogels include, but are not limited to, agar, agarose, alginic acid, alguronic acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan polysaccharides (e.g., of the kappa, iota or lambda variety), cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, polysaccharide, levan, fucoidan, galactoglucomannan, galactomannans, galactosaminogalactans, gellan gum, dextran, glucomannan, glucorun The polysaccharides described herein can be oxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentamer starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scissoran, sizoran, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof. As described throughout, the polysaccharide can be further functionalized depending on the desired cross-linking chemistry and / or additional comonomers employed in the hydrogel. For example, in one embodiment, one or more polysaccharides described herein are functionalized with acrylate or acrylamide.

[0117] In one embodiment, a single hydrogel particle or a plurality of hydrogel particles comprises a peptide, a protein, a protein domain or a combination thereof as a hydrogel monomer or a plurality of hydrogel monomers. In a further embodiment, the protein is a structural protein or its domain, such as, for example, silk protein, elastin, myomegalin or collagen or its domain. In one embodiment, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In an even further embodiment, the structural protein is collagen. In yet a further embodiment, the collagen is type I collagen, type II collagen or type III collagen or a combination thereof. In another embodiment, the hydrogel monomer comprises a proteoglycan. In a further embodiment, the proteoglycan is decorin, biglycan, testisan, bikunitz inhibitor, fibromodulin, lumican or its domain.

[0118] In another embodiment, acrylate-functionalized structural protein hydrogel monomers (e.g., acrylate-functionalized proteins or protein domains, such as silk protein, elastin, myomegalin, collagen, proteoglycans, or functionalized domains thereof) are used as components of the hydrogels provided herein. In further embodiments, acrylate-functionalized structural protein hydrogel monomers include proteoglycans, such as decorin, biglycan, testoglycan, bikunitz inhibitor, fibromodulin, lumican, or domains thereof.

[0119] In one embodiment, PEG monomers and oligopeptides that can simulate extracellular matrix proteins are used in the hydrogels provided herein, such as multi-arm PEG, integrin binding peptides and double cysteine ​​matrix metalloproteinase peptides functionalized with vinyl sulfone, such as Lutolf et al. (2003). Proc. Natl. Acad. Sci. USA 100, 5413-5418, which are incorporated by reference in their entirety for all purposes. In this particular embodiment, the hydrogel is formed by a Michael-type addition reaction between dithiolated oligopeptides and the vinyl sulfone groups on PEG. The scope of other compatible chemicals that can be incorporated here is clear to those skilled in the art, and follows the general chemical reactivity principle, see, for example, Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools. lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf).

[0120] Other biologically active domains in natural proteins can also be used as hydrogel monomers or parts thereof. For example, cell adhesion integrin binding domains, controlled release affinity binding domains or transglutaminase cross-linking domains can be used in the hydrogels provided herein. Details for producing such hydrogels can be found in the following documents: Martino et al. (2009). Biomaterials 30, 1089; Martino et al. (2011). Sci. Trans. Med. 3, 100ra89; Hu and Messersmith (2003). J. Am. Chem. Soc. 125, 14298, each of which is incorporated by reference in its entirety for all purposes.

[0121] In one embodiment, a recombinant DNA method is used to produce a protein that is designed to gel in response to changes in pH or temperature, such as by the method described in Petka et al. (1998). Science 281, pp. 389-392, which is incorporated by reference in its entirety for all purposes. In brief, the protein consists of a terminal leucine zipper domain flanked by a water-soluble polyelectrolyte segment. In near-neutral aqueous solution, the coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

[0122] A further range of biocompatible monomers that may be incorporated are known in the art, see for example the non-degradable biocompatible monomers disclosed in Shastri (2003). Current Pharmaceutical Biotechnology 4, pp. 331-337, which is incorporated herein by reference in its entirety for all purposes. Other monomers are provided in the following documents: de Moraes Porto (2012). Polymer Biocompatibility, Polymerization, Dr. Ailton De Souza Gomes (ed.), ISBN: 978-953-51-0745-3; InTech, DOI: 10.5772 / 47786; Heller et al. (2010). Journal of Polymer Science Part A: Polymer Chemistry 49, pp. 650-661; Final Report for Biocompatible Materials (2004), The Board of the Biocompatible Materials and the Molecular Engineering in Polymer Science programs, ISBN 91-631-4985-0, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0123] In an embodiment, the polymerized monomer is a homopolymer. The term "homopolymer" refers to a polymerized monomer containing a single repeating monomer unit. In an embodiment, the polymerized monomer is a copolymer. The term "copolymer" refers to a polymer prepared by the reaction of two different monomers. The two different monomers are referred to as comonomers. In an embodiment, the monomer is a difunctional monomer. In an embodiment, one of the comonomers is a difunctional monomer. In an embodiment, both comonomers are difunctional monomers.

[0124] In an embodiment, the hydrogel is synthesized in the presence of a cross-linking agent. In an embodiment, the cross-linking agent is selected from any one of the following: ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, and N,N'-15 methylenebisacrylamide.

[0125] In embodiments, the hydrogel is synthesized in the presence of a polymerization initiator. In embodiments, the polymerization initiator is a persulfate or an equivalent initiator that catalyzes the formation of free radicals. In embodiments, the persulfate is any water-soluble persulfate. Non-limiting examples of water-soluble persulfates include ammonium persulfate and alkali metal persulfates. In embodiments, the alkali metal persulfate is a lithium, sodium or potassium alkali metal persulfate. In embodiments, the persulfate is ammonium persulfate or potassium persulfate. In further embodiments, the polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

[0126] In embodiments, the polymerization of the hydrogel can be accelerated by an accelerator that can catalyze the formation of unstable chemical side groups. In embodiments, the accelerator is a tertiary amine. In embodiments, the tertiary amine is a water-soluble tertiary amine. In embodiments, the accelerator is N,N,N',N'-tetramethylethylenediamine, 3-(dimethylamino)propionitrile or N,N,N',N'-tetramethylethylenediamine (TEMED). In embodiments, the accelerator is azobis(isobutyronitrile) (AIBN).

[0127] In embodiments, the amount of monomer can be varied, for example, to obtain specific optical or morphological properties that are substantially similar to target cells. In embodiments, the monomer is present at about 10% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the monomer is present in an amount of about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 143%, about 144%, about 145%, about 146%, about 147%, about 148%, about 149%, about 200%, about

[0128] In embodiments, the polymerized monomer is present at about 10% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the polymerized monomer is present at about 15% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the polymerized monomer is present at about 20% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the polymerized monomer is present in an amount of about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about %, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, and all values ​​and subranges therebetween, inclusive.

[0129] In an embodiment, the difunctional monomer is present at about 10% by weight of the hydrogel to about 95% by weight of the hydrogel. In an embodiment, the difunctional monomer is present at about 15% by weight of the hydrogel to about 95% by weight of the hydrogel. In an embodiment, the difunctional monomer is present at about 20% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the difunctional monomer is present in an amount of about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about About 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, and all values ​​and subranges therebetween, inclusive.

[0130] In embodiments, the comonomer is present at about 10% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the comonomer is present at about 15% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the comonomer is present at about 20% by weight of the hydrogel to about 95% by weight of the hydrogel. In embodiments, the comonomer is present in an amount of about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about %, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, and all values ​​and subranges therebetween, inclusive.

[0131] In an embodiment, the monomer is selected from any one of the following: 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glyceryl methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate.

[0132] In an embodiment, the monomer is selected from any one of the following: lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinyl pyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glyceryl methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, derivatives thereof, or combinations thereof.

[0133] In an embodiment, the monomer is selected from any of the following: phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate , N-phenylacrylamide, N-phenylmethacrylamide, N-benzylacrylamide, N-benzylmethacrylamide, N,N-dibenzylacrylamide, N,N-dibenzylmethacrylamide, N-dibenzylmethylacrylamide, N-(4-methylphenyl)methacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-tritylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N, N-phenylphenethyl acrylamide, N-benzhydryl methacrylamide, N-(4-methylphenyl)methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl) methacrylamide, N-trityl methacrylamide, N-(4-hydroxyphenyl) methacrylamide, N,N-methylphenyl methacrylamide, N,N'-phenylphenethyl methacrylamide, N-vinyl carbazole, 4-vinyl pyridine or 2-vinyl pyridine.

[0134] In an embodiment, the monomer is selected from any of the monomers disclosed in US Pat. No. 6,657,030, which is incorporated herein by reference in its entirety for all purposes.

[0135] In embodiments, the monomer is a synthetic monomer. In embodiments, the monomer is a biological monomer.

[0136] In an embodiment, the monomer is selected from any of the monomers in the Thermo Scientific Crosslinking Technical Handbook entitled "Easy molecular bonding crosslinking technology" (available at tools.lifetechnologies.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf), the disclosure of which is incorporated by reference in its entirety for all purposes.

[0137] In embodiments, the hydrogel is a synthetic hydrogel. The synthetic hydrogel comprises synthetic monomers. In embodiments, the hydrogel is a biohydrogel. The biohydrogel comprises biomolecules, such as peptides, proteins, monosaccharides, disaccharides, polysaccharides or carbohydrates. In embodiments, the biohydrogel comprises functional groups found on biomolecules, such as primary amines, sulfhydryls, carbonyls, carboxylic acids or carbohydrates. In embodiments, the hydrogel is a hybrid hydrogel. The hybrid hydrogel comprises synthetic components and biomolecules and / or functional groups found on biomolecules. In embodiments, proteins, peptides or carbohydrates can be used as separate monomers to form a hydrogel that includes or does not include synthetic monomers (or polymers) and is combined with chemically compatible comonomers and crosslinking chemicals. Compatible crosslinking chemicals include but are not limited to amines, carboxyls and other reactive chemical side groups.

[0138] In embodiments, one or more of monomer, comonomer, difunctional monomer is bis / acrylamide in various cross-linking ratios. In embodiments, one or more of monomer, comonomer, difunctional monomer includes allylamine. In embodiments, in embodiments, one or more of monomer, comonomer, difunctional monomer provides for the chemical functional group of secondary labeling / conjugation. In embodiments, one or more of monomer, comonomer, difunctional monomer includes alginate. For example, hydrazine (for example, with NHS ester compound) or EDC coupling reaction (for example, with maleimide compound) can be used to build the hydrogel of the present disclosure.

[0139] In an embodiment, the polymerized monomer is a biodegradable monomer. In an embodiment, the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein or a protein domain. In an embodiment, the biodegradable monomer is a structural polysaccharide. In an embodiment, the biodegradable monomer is selected from agar, agarose, alginic acid, polysaccharide acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysaccharide, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, dextran , glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan and combinations thereof.

[0140] In embodiments, the hydrogel is biodegradable. In embodiments, the hydrogel comprises a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. In embodiments, the hydrogel comprises a structural polysaccharide. In embodiments, the hydrogel comprises agar, agarose, alginic acid, polysaccharide acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, chrysophyllin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysaccharide, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, dextran, glucomannan, Dextran, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof.

[0141] In embodiments, the hydrogel beads include biodegradable polymers as monomers. In embodiments, biodegradable polymers are poly (esters) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), polylactic acid co-glycolic acid (PLGA) and copolymers thereof. These polymers can be degraded by hydrolysis, thereby dissolving macromolecular particles. In embodiments, biodegradable polymers are carbohydrates or proteins or combinations thereof. In one embodiment, monosaccharides, disaccharides or polysaccharides (e.g., glucose, sucrose or maltodextrin), peptides, proteins (or their domains) are used as hydrogel monomers. In embodiments, biodegradable polymers are poly (hydroxyalkanoates) of PHB-PHV categories, other poly (esters) or natural polymers, such as modified poly (sugars), such as starch, cellulose and chitosan. In embodiments, biocompatible polymers are adhesive proteins, cellulose, carbohydrates, starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), dextran, lignin, polyamino acids, amino acids or chitin. Such biodegradable polymers are commercially available, for example, from Sigma Aldrich (St. Louis, MO).

[0142] In an embodiment, the protein monomer comprises only natural amino acids. In an embodiment, the protein monomer comprises non-natural amino acids. For example, self-assembling artificial proteins and proteins with non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or introduced by synthetic methods, see, for example, Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587, the disclosure of which is incorporated by reference in its entirety for all purposes) or its protein domains can also be used as hydrogel monomers. The scope of non-natural (non-natural or unnatural) amino acids that can be incorporated into such compositions is well known to those skilled in the art (Zhang et al. (2013). Current Opinion in Structural Biology 23, pp. 581-587; it is incorporated by reference in its entirety for all purposes). In an embodiment, a biodegradable polymer is used as a comonomer. In an embodiment, in one embodiment, the biodegradable polymer is a difunctional monomer.

[0143] In an embodiment, the hydrogel beads described herein comprise a degradable polymer as a monomer. In an embodiment, the degradable polymer is a poly(ester) based on PLA, PGA, PCL, PLGA and copolymers thereof. In an embodiment, the degradable polymer is based on any of the monomers described herein, which may be degraded by mechanical degradation, chemical degradation and combinations thereof or by any other degradation mechanism. For example, the monomer may be acrylamide, and the degradable polymer formed therefrom may be degraded by exposure to potassium persulfate.

[0144] In embodiments, degradation of a single hydrogel particle or a plurality of hydrogel particles (whether by biodegradable means, lysis, or otherwise) can result in the release of a substance contained therein. For example, when the substance is a biomolecule such as hemoglobin, degradation of hydrogel particles comprising hemoglobin can allow for measurement of hemoglobin separated from hydrogel monomers. In another instance, when the substance is a biomolecule such as hemoglobin, degradation of hydrogel particles comprising hemoglobin can be performed by a lysis buffer.

[0145] In embodiments, the concentration of the difunctional monomer and / or polymerized monomer may be adjusted to alter the refractive index of the hydrogel.

[0146] In an embodiment, the hydrogel beads described herein can be cracked. The cleavable synthetic beads of the present invention allow users to measure both complete beads and cracked beads. In an embodiment, the hydrogel beads are cracked with a lysis buffer. In an embodiment, the lysis buffer is ammonium chloride. The blood lysis buffer generally uses ammonium chloride, including 1X RBC lysis buffer and 10X RBC lysis buffer from ThermoFisher Scientific. The blood lysis buffer for clinical blood samples is designed to crack anucleated red blood cells and preserve white blood cells to perform quantitative measurements of white blood cell counts and hemoglobin. Other lysis buffers can be designed to dissolve engineered particles, including strong reducing agents such as dithiothreitol (DTT) or beta mercaptoethanol (BME). Other non-limiting examples include divalent ions such as ethylenediaminetetraacetic acid (EDTA) or citrate.

[0147] In an embodiment, the hydrogel beads are formed by precipitation polymerization, as described in Elbert (2011), Acta Biomater.7, pp. 31-56. This reference is incorporated herein by reference in its entirety for all purposes. Precipitation polymerization is a technique for producing microparticles using the solubility difference between monomers and polymers. Specifically, it is known that larger polymer chains generally have lower solubility than smaller polymer chains. Therefore, above a specific molecular weight, phase separation can be advantageous. Precipitation polymerization initially starts with solution polymerization in a single isomeric system. In one embodiment, shortly after the start of polymerization, there is a relatively high concentration of polymer chains, which is conducive to phase separation by nucleation. As the polymerization proceeds, the concentration of polymer chains is low, and existing particles capture the chains before new particle nucleation can occur. Therefore, the nucleation of particles occurs only in a short period of time shortly after the reaction begins, which results in a narrow size distribution of particles in one embodiment. In an embodiment, the hydrogel beads can be formed by one or more methods selected from: photolithographic particle formation (Helgeson et al. (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106-117, which is incorporated herein by reference in its entirety for all purposes), membrane emulsification (e.g., by the microsieve emulsification technique described by Nanomi BV (The Netherlands)), microchannel emulsification (Sugiura et al. (2002). Languimir 18, pp. 5708-5712, which is incorporated herein by reference in its entirety), or bulk emulsification (SNF Floerger, available at snf.com.au / downloads / Emulsion_Handbook_E.pdf, which is incorporated herein by reference in its entirety).

[0148] In embodiments, hydrogel beads are formed in a microfluidic device having two oil channels that are focused on a central stream of an aqueous monomer solution. In embodiments, droplets are formed at the interface of the two channels and the central stream so that the droplets break apart in an oil-in-water emulsion. In embodiments, after the droplets are formed, they are stabilized prior to polymerization. In embodiments, the droplets are stabilized by adding a surfactant to the oil phase. In embodiments, the droplets are not stabilized prior to polymerization. In embodiments, polymerization of the monomers is triggered by adding an accelerator (e.g., N,N,N',N'-tetramethylethylenediamine) to one or both oil channels after the initial droplets are formed.

[0149] In an embodiment, the monomer aqueous solution as provided above may include a single monomer species or multiple monomer species. In an embodiment, the monomer aqueous solution includes a comonomer, a difunctional monomer, or a combination thereof. In an embodiment, the monomer or monomers include a difunctional monomer. In an embodiment, the monomer is one of the above monomers. In an embodiment, a comonomer is used to adjust forward scattering or side scattering. In an embodiment, a comonomer is used to adjust the refractive index of the hydrogel beads.

[0150] In an embodiment, the center stream of the aqueous monomer solution comprises a crosslinker. In an embodiment, the crosslinker is N,N'-bisacrylamide. In an embodiment, the center stream of the aqueous monomer solution comprises a crosslinker, an accelerator, and the monomer. In an embodiment, the aqueous monomer solution comprises an initiator. In an embodiment, the initiator is an oxidant. In an embodiment, the oxidant is ammonium persulfate.

[0151] In an embodiment, the hydrogel beads are produced by polymerizing droplets. Microfluidic methods for producing a plurality of droplets (including fluid droplets and hardened droplets) are described in U.S. Patent Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each of which is incorporated herein by reference in its entirety for all purposes. Such methods provide a plurality of droplets containing a first fluid and substantially surrounded by a second fluid, wherein the first fluid and the second fluid are substantially immiscible (e.g., a droplet containing a water-based liquid substantially surrounded by an oil-based liquid). IV-B. Pro-apoptotic Signals and Pro-apoptotic Signal Binding Agents

[0152] In embodiments, the hydrogel beads described herein include a pre-apoptotic signal or a pre-apoptotic signal binding agent. In embodiments, the pre-apoptotic signal binding agent is present on cells undergoing apoptosis and binds to the pre-apoptotic signal. In embodiments, the hydrogel beads comprising the pre-apoptotic signal binding agent serve as a mimic of apoptotic cells. In some embodiments, the pre-apoptotic signal binding agent is artificially designed to bind to a known pre-apoptotic signal (e.g., an antibody targeting a pre-apoptotic signal).

[0153] Table 1A includes pro-apoptotic signal binding agents and the pro-apoptotic signals to which they bind. In embodiments, the hydrogel beads described herein include one, two, three, four, five, six, seven, eight, nine or ten (including all ranges and subranges therebetween) different pro-apoptotic signal binding agents. In embodiments, the hydrogel beads described herein include one, two, three, four, five, six, seven, eight, nine or ten (including all ranges and subranges therebetween) different pro-apoptotic signals. Table 1A

[0154] The following patent documents and publications describe pro-apoptotic signal binders and pro-apoptotic signals and are incorporated herein by reference in their entirety for all purposes: Elmore, Toxicol Pathol. 2007; 35(4):495–516; Armstrong et al. EMBO Rep. 2011 Apr 1; ​​12(4):287–288; and U.S. Publication No. 2022 / 0143160.

[0155] In embodiments, the pro-apoptotic signal comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to Annexin V (UniProt Accession No. P08758), a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to SEQ ID NO: 103, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to an apo-15 peptide, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to β2-glycoprotein 1 or a fragment thereof (UniProt Accession No. D9IWP9) (e.g., domain V). , at least 95%, at least 98% or 100% identical to SEQ ID NO: 1, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to milk fat globule-EGF-factor 8 (MFG-E8) (UniProt Accession No. Q08431), a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to a phosphatidylserine receptor or a fragment thereof, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 1 ID NO: 102 having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to CD36 (UniProt Accession No. P16671), a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to LDL receptor-related protein (UniProt Accession No. P01130), or a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to an anti-calreticulin antibody or an antigen-binding fragment thereof.

[0156] In embodiments, the pro-apoptotic signal comprises Annexin V. Annexin V binds to phosphatidylserine present on the outer leaflet of the plasma membrane on apoptotic cells. In an embodiment, the annexin V is from a species selected from the group consisting of: human, rabbit, mouse, giant panda (Ailuropoda melanoleuca), Peruvian night monkey (Aotus nancymaae), minke whale (Balaenoptera acutorostrata scammoni), blue whale (Balaenoptera musculus), zebu (Bosindicus), cattle (Bos taurus), zebu, yak (Bos mutus), cattle, water buffalo (Bubalus bubalis), common marmoset (Callithrix jacchus), Bactrian camel (Camelus bactrianus), Chinese pastoral dog (Canis lupusfamiliaris), goat (Capra hircus), Philippine tarsier (Carlito syrichta), American beaver (Castorcanadensis), cattle, Panamanian capuchin (Cebus imitator), Canadian elk (Cervus canadensis), red deer (Cervus elaphus), Tarim Hara red deer (Cervus hanglu yarkandensis), Beluga whale (Delphinapterus leucas), Osman's kangaroo rat (Dipodomys ordii), Desert flag-tailed kangaroo rat (Dipodomys spectabilis), Indian elephant (Elephas maximus indicus), Przewalski's horse (Equusprzewalskii), Gray whale (Eschrichtius robustus), Domestic cat (Felis catus), Western gorilla (Gorillagorilla), Eastern gorilla (Gorilla beringer), Mink (Gulo gulo luscus), Gray seal (Halichoerus grypus), Striped hyena (Hyaena hyaena), Silvery gibbon (Hylobates moloch), Multi-striped ground squirrel (Ictidomys tridecemlineatus), African jerboa (Jaculus jaculus), Pacific short-beaked dolphin (Lagenorhynchus obliquidens), ring-tailed lemur (Lemur catta), white-fin dolphin (Lipotesvexillifer), African savanna elephant (Loxodonta africana), long-tailed macaque (Macacafascicularis), macaque (Macaca mulatta), drill (Mandrillus leucophaeus), yellow-bellied marmot (Marmota flaviventris), Alpine marmot (Marmota marmota marmota), American marmot (Marmota monax), forest musk deer (Moschusberezovskii), barking deer (Muntiacus muntjak), pet mink (Mustela putorius furo), American water mink (Neogale vison), Hawaiian monk seal (Neomonachus schauinslandi), white-cheeked gibbon (Nomascusleucogenys), raccoon dog (Nyctereutes procyonoides), Pacific walrus (Odobenus rosmarusdivergens), Texas subspecies of white-tailed deer (Odocoileus virginianus texanus), killer whale (Orcinusorca), sheep (Ovis aries), chimpanzee (Pan troglodytes), East African baboon (Papio anubis), Pacific pocket mouse (Perognathus longimembris pacificus), harbor seal (Phoca vitulina), sperm whale (Physeter catodon), Ugandan red colobus (Piliocolobus tephrosceles), Clarix's sifaka (Propithecus coquereli), Svalbard reindeer (Rangifer tarandus platyrhyncus), Yunnan snub-nosed monkey (Rhinopithecus bieti), Amazon squirrel monkey (Saimiri boliviensis boliviensis), Eastern American squirrel (Sciurus carolinensis), shrew (Sorex araneus), Eurasian wild boar (Sus scrofa), black leaf monkey (Trachypithecus francoisi), tree shrew (Tupaia chinensis), bottlenose dolphin (Tursiopstruncatus), Arctic ground squirrel (Urocitellus parryii), polar bear (Ursus maritimus), Arctic fox (Vulpes lagopus) and California sea lion (Zalophus californianus). In an embodiment, the annexin V protein is related to the protein having SEQ IDThe annexin V protein of any one of NO:1-101 has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity.

[0157] In embodiments, the pro-apoptotic signal comprises a polypeptide of SEQ ID NO: 103. In embodiments, the polypeptide of SEQ ID NO: 103 is cyclic. In embodiments, the pro-apoptotic signal comprises an apo-15 peptide. Apo-15 is a cyclic peptide having an amino acid sequence of GRKKWFW (SEQ ID NO: 104). The structure of the apo-15 peptide is described in Barth et al. Nat Commun. 2020 Aug 12; 11(1): 4027, which is incorporated herein by reference in its entirety for all purposes. In embodiments, the cyclic apo-15 peptide comprises any fluorescent dye described herein. In embodiments, the dye is conjugated to one or more of the tryptophan residues of SEQ ID NO: 104. In embodiments, the dye is BODIPY. In embodiments, the fluorescent dye is Trp-BODIPY. The Trp-BODIPY fluorophore is described in Mendive et al. Nat Protocols. 2017; 12: 1588-1619, which is incorporated herein by reference in its entirety for all purposes.

[0158] In embodiments, the pre-apoptotic signal is a phospholipid binding protein. In embodiments, the phospholipid binding protein is β2-glycoprotein 1 (β2GPI) or prothrombin. Both proteins bind to phosphatidylserine on apoptotic cells. In embodiments, the pre-apoptotic signal is domain V of β2GPI. β2GPI and prothrombin are described in the following documents: McDonnell et al. Blood Rev. January 2020; 39: 100610 and Houston et al. Mol Cell Biochem. February 2011; 348 (1-2): 109-15. These references are incorporated herein by reference in their entirety for all purposes.

[0159] In an embodiment, the pro-apoptotic signal is milk fat globule-EGF-factor 8 (MFG-E8). MFG-E8 is an anti-inflammatory glycoprotein that mediates the clearance of apoptotic cells. The following references describe MFG-E8 and are incorporated herein by reference in their entirety for all purposes: Lauber et al. Cell Death Differ. 2013 Sep; 20(9): 1230-40 and Borishenko et al. Cell Death Differ. 2004 Aug; 11(8): 943-5.

[0160] In embodiments, the pro-apoptotic signal is a phosphatidylserine receptor or a fragment thereof. Phosphatidylserine receptors are expressed by phagocytes. There are a variety of phosphatidylserine receptors with different structures, cell type expressions, and abilities to bind to phosphatidylserine. The following references describe phosphatidylserine receptors and are incorporated herein by reference in their entirety for all purposes: Naeini et al. Cell Mol Biol Lett. 2020 Mar 26; 25:23. In an embodiment, the phosphatidylserine receptor is selected from any one of the following: brain-specific angiogenesis inhibitor-1 (Bai1), Axl, Tyro3, ​​Mer, TIM-1 (also known as "KIM-1"), TIM-4, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), stabilin-1, stabilin-2, CD300a, CD300b, CD300f, receptor for advanced glycation end products (RAGE), complement component 1q (C1q), β2-glycoprotein I (β2GPI), annexin or integrin αVβ3 / β5. Table 1B contains the UniProt accession numbers of the above-mentioned phosphatidylserine receptors. In an embodiment, the pro-apoptotic signal is a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity with any one of the phosphatidylserine receptors of Table 1B. Table 1B

[0161] In an embodiment, the pro-apoptotic signal is CD36. CD36 binds to thrombospondin-1 expressed on apoptotic cells. The following article describes the role of CD36 in apoptosis and is incorporated herein by reference in its entirety for all purposes: Fadok et al. J Immunol. 1998 Dec 1; 161(11): 6250-7.

[0162] In an embodiment, the pro-apoptotic signal comprises an LDL receptor-associated protein. The LDL receptor-associated protein binds to calreticulin and triggers the clearance of apoptotic cells. The following article describes the role of LDL receptor-associated protein in apoptosis and is incorporated herein by reference in its entirety: Gardai et al. Cell. 2005 Oct 21: 123(2): 321-324. In an embodiment, the pro-apoptotic signal is an anti-calreticulin antibody or an antigen-binding fragment thereof.

[0163] In embodiments, any of the pro-apoptotic signals described herein may comprise a fluorescent dye. In embodiments, the fluorescent dye is any fluorescent dye described herein.

[0164] In an embodiment, the pro-apoptotic signal binding agent is selected from phosphatidylserine, anti-annexin V antibody or an antigen-binding fragment thereof, annexin I, calreticulin, anti-CD36 antibody or an antigen-binding fragment thereof, thrombospondin-1 (TSP-1), anti-β2-glycoprotein I antibody or an antigen-binding fragment thereof, anti-milk fat globule-EGF-factor 8 (MFG-E8) or an antigen-binding fragment thereof, anti-phosphatidylserine receptor or an antigen-binding fragment thereof, or anti-LDL receptor-related protein or an antigen-binding fragment thereof.

[0165] In an embodiment, the pro-apoptotic signal binding agent is an anti-Annexin V antibody or an antigen-binding fragment thereof. In an embodiment, the anti-Annexin V antibody or an antigen-binding fragment thereof binds to SRLYDAYELKHALKG (SEQ ID NO: 102) of the Annexin V protein or a fragment thereof. In an embodiment, the anti-Annexin V antibody can be purchased from Boster Biological Technology (#PA1008). In an embodiment, the anti-Annexin V antibody or an antigen-binding fragment thereof binds to an Annexin V protein that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 1-101. In embodiments, the anti-Annexin V antibody or antigen-binding fragment thereof binds to an Annexin V protein from a species selected from any one of the following: human, rabbit, mouse, giant panda, Peruvian night monkey, minke whale, blue whale, zebu x Bostaurus, zebu, yak, cattle, buffalo, common marmoset, Bactrian camel, Chinese pastoral dog, goat, Philippine tarsier, American beaver, cattle, Panamanian capuchin, Canadian elk, red deer, Tarim Hara elk, beluga whale, Osso's kangaroo rat, desert flag-tailed kangaroo rat, Indian elephant, Przewalski's horse, gray whale, domestic cat, western gorilla, eastern gorilla, wolverine, gray seal, Homo sapiens sapiens), striped hyena, silvery gibbon, multi-striped ground squirrel, African jerboa, Pacific short-necked dolphin, ring-tailed lemur, white-fin dolphin, African savanna elephant, long-tailed macaque, macaque, drill baboon, yellow-bellied marmot, Alpine marmot, American marmot, forest musk deer, barking deer, pet mink, American water mink, Hawaiian monk seal, white-cheeked gibbon, raccoon dog, Pacific walrus, white-tailed deer texas subspecies, killer whale, sheep, chimpanzee, East African baboon, Pacific pocket mouse, harbor seal, sperm whale, Ugandan red colobus, Crow's crested lemur, Svalbard reindeer, Yunnan snub-nosed monkey, Amazon squirrel monkey, Eastern American squirrel, shrew, Eurasian wild boar, dark leaf monkey, tree shrew, bottlenose dolphin, Arctic ground squirrel, polar bear, Arctic fox and California sea lion.

[0166] In an embodiment, the pro-apoptotic signal binding agent is phosphatidylserine. The structure of phosphatidylserine comprises a glycerol backbone connected to two fatty acids and a phosphoserine molecule. The structure of phosphatidylserine is as follows. The R group in the structure represents a fatty acid.

[0167] In an embodiment, the pro-apoptotic signal binding agent is annexin I. Annexin I is a glucocorticoid-regulated protein that is involved in the regulation of phagocytosis, cell signaling, and proliferation, and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Annexin I expression is elevated in apoptotic cells and appears to play a role in bridging phosphatidylserine on apoptotic cells to phagocytes and enhancing the recognition of apoptotic cells by phagocytes, such as macrophages.

[0168] In embodiments, any of the pro-apoptotic signal binding agents described herein may comprise a fluorescent dye. In embodiments, the fluorescent dye is any fluorescent dye described herein.

[0169] In some embodiments, the pre-apoptotic signal binding agent or the pre-apoptotic signal is embedded in the hydrogel matrix. In some embodiments, the pre-apoptotic signal binding agent or the pre-apoptotic signal is attached to the surface of the hydrogel. In some embodiments, the pre-apoptotic signal binding agent or the pre-apoptotic signal is attached to the hydrogel via a functional group (e.g., an amine group or biotin or streptavidin). IV-C. Encapsulated Nucleic Acids

[0170] In an embodiment, the hydrogel beads described herein further comprise encapsulated nucleic acids. The hydrogel beads comprising encapsulated nucleic acids act as a mimic of dead cells. Dead cells typically have damaged cell membranes and thus exposed DNA.

[0171] In embodiments, the hydrogel beads described herein comprise (a) polymerized monomers and bifunctional monomers; (b) a pro-apoptotic signal; and (c) an encapsulated nucleic acid.

[0172] In embodiments, the hydrogel beads described herein comprise (a) polymerized monomers and bifunctional monomers; (b) a pro-apoptotic signal binding agent; and (c) an encapsulated nucleic acid.

[0173] In embodiments, the encapsulated nucleic acid is selected from any one of the following: double-stranded DNA, single-stranded DNA, complementary DNA (cDNA) and RNA. In embodiments, the encapsulated nucleic acid is double-stranded DNA.

[0174] In an embodiment, the hydrogel beads comprising the encapsulated nucleic acid are bound to a dye. In an embodiment, the dye is intercalated between DNA or RNA bases. In an embodiment, the dye binds to the major groove of DNA. In an embodiment, the dye binds to the minor groove of DNA. In an embodiment, the dye is selected from 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), DRAQ5 TM 、DRAQ7TM 、CytoPhase TM Violet, Helix NP TM Blue, Helix NP TM Green, Helix NP TM NIR、YOYO TM -1.TOTO TM -1 iodide (Thermo Fisher Scientific), SYTOX TM Blue, ethidium bromide, SYBR TM Gold, SYBR TM Green, SYBR TM Safe, and crystal violet.

[0175] Those skilled in the art will be familiar with other dyes and detectors for DNA which, in some embodiments, are compatible with the invention of the present disclosure. IV-D. Additional components

[0176] In an embodiment, the hydrogel beads described herein may include one or more additional chemical moieties. In an embodiment, the hydrogel beads described herein include one or more fluorescent dyes. In an embodiment, the fluorescent dye is attached to the hydrogel beads via a covalent bond. In an embodiment, the fluorescent dye is attached to the hydrogel beads via a non-covalent interaction. In an embodiment, the fluorescent dye is selected from one or more of the following: 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein succinimidyl ester; 5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein; 5-(and-6)-carboxyfluorescein; S-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl) ether, -alanine-carboxamide or succinimidyl ester; 5-carboxyfluorescein succinimidyl ester; 6-carboxyfluorescein succinimidyl ester; Amine esters; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2',7'-difluorofluorescein; eosin-5-isothiocyanate; erythrosine-5-isothiocyanate; 6-(fluorescein-5-formamido)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-formamido)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; 488 Carboxylic acid or succinimide ester; Oregon 488 Isothiocyanate; Oregon 488-X Succinimidyl ester; Oregon 500 Carboxylic acid; Oregon 500 Carboxylic acid, succinimide ester or triethylammonium salt; Oregon 514 Carboxylic acid; Oregon 514 Carboxylic acid or succinimide ester; Rhodamine Green TM Carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green TM Carboxylic acid, trifluoroacetamide, or succinimidyl ester; Rhodamine Green TM -X succinimidyl ester or hydrochloride; RhodolGreen TM Carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl)sulfone rhodamine or bis(succinimidyl ester); 5-(and-6)carboxynaphthofluorescein, 5-(and-6)carboxynaphthofluorescein succinimidyl ester; 5-carboxyrhodamine 6G hydrochloride; 6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidyl ester; 6-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-(and-6)-carboxyrhodamine 6G succinimidyl ester; 5-carboxy-2',4',5',7'-tetrabromosulfonefluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt ; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimide ester; 6-carboxytetramethylrhodamine succinimide ester; 5-(and-6)-carboxytetramethylrhodamine succinimide ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimide ester; 6-carboxy-X-rhodamine succinimide ester; 5-(and-6)-carboxy-X-rhodamine succinimide ester; 5-carboxy-X-rhodamine triethylammonium salt; LissamineTM rhodamine B sulfonyl chloride; malachite green; isothiocyanate; Mono(sulfosuccinimide ester); 21 carboxylic acid or succinimide ester; 7 carboxylic acid or succinimide ester; Rhodamine RedTM-X succinimide ester; 6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimide ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Sulfonyl; Texas Sulfonyl chloride; Texas -XSTP ester or sodium salt; Texas -X succinimidyl ester; Texas -X succinimidyl ester; and X-rhodamine-5-(and -6) isothiocyanate, commercially available from Invitrogen Dyes, including but not limited to FL; TMR STP ester; TR-X STP ester; 630 / 650-X STP ester; 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid succinimide ester; 4,4-difluoro-4-bora-3a,4a-diaza-sym-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3-pentanoic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sym-indacene-3-propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4 a-diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; sulfosuccinimide ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-indacene-3-propionyl)sulfoalanine, succinimide ester or triethyl ammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-indacene-3-propionic acid; succinimide ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-indacene-3-propionyl)amino)hexanoic acid or succinimide ester; 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid succinimide ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid succinimide ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-symmetric indacene-3-yl)phenylvinyloxy)acetyl)aminohexanoic acid or succinimide ester; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid; 4,4-difluoro-5-phenylvinyl-4-bora-3a,4a-diaza-symmetric indacene-3-propionic acid; succinimide ester;4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-indacene-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-indacene-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacene-3-propionic acid succinimidyl ester; 6-(((4-( 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimide ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-indacen-3-yl)phenyloxy)acetyl)amino)hexanoic acid or succinimide ester, Alexa fluor dyes commercially available from Invitrogen, including but not limited to Alexa; 350 Carboxylic acid; Alexa 430 Carboxylic acid; Alexa 488 Carboxylic acid; Alexa 532 Carboxylic acid; Alexa 546 Carboxylic acid; Alexa 555 Carboxylic acid; Alexa 568 Carboxylic acid; Alexa 594 Carboxylic acid; Alexa 633 Carboxylic acid; Alexa 64 7 carboxylic acid; Alexa 660 carboxylic acid; and Alexa 680 carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech, include but are not limited to Cy3 NHS ester; Cy 5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester.

[0177] In embodiments, the hydrogel beads may comprise from 1 to about 20 fluorescent dyes, from 1 to about 10 fluorescent dyes, or from 1 to about 5 fluorescent dyes. In embodiments, the hydrogel beads comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 (including all values ​​and subranges therebetween, inclusive) fluorescent dyes.

[0178] In embodiments, the hydrogel beads comprise "rainbow particles". The rainbow particles contain a mixture of fluorophores. In embodiments, the rainbow particles comprise from 1 to about 20 fluorophores, from 1 to about 10 fluorophores, or from 1 to about 5 fluorophores. In embodiments, the hydrogel beads comprise rainbow particles having 1, 2, 3, 4, 5, 6, 7, 8, 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 (including all values ​​and subranges therebetween, inclusive) fluorophores. In embodiments, the user selects the wavelength used to excite the rainbow particles, depending on the fluorophore being interrogated. Rainbow particles are commercially available, for example from BD Biosciences (catalog numbers 556298 (mid-range FL1 fluorescence), 556286 (6 colors, 3.0-3.4 μm), 556288 (6 colors, 6.0-6.4 μm), 559123 (8 colors)) and Spherotech (in various diameters, for example, catalog numbers RCP20-5 (4 colors), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)).

[0179] In an embodiment, the hydrogel beads comprise a scattering modulating additive. In an embodiment, the scattering modulating additive comprises polymer nanoparticles. In an embodiment, the polymer nanoparticles comprise polystyrene. In an embodiment, the scattering modulating additive comprises a comonomer. In an embodiment, the scattering modulating additive comprises a suspension of nanoparticles.

[0180] In embodiments, the hydrogel beads are chemically functionalized hydrogel particles. In embodiments, the hydrogel comprises free amine groups. In embodiments, the pro-apoptotic signal binding agent is attached to the free amine groups. In embodiments, the pro-apoptotic signal is attached to the free amine groups. In embodiments, the hydrogel beads comprise allylamine. In embodiments, the hydrogel beads comprise biotin. In embodiments, the hydrogel beads comprise streptavidin. In embodiments, the hydrogel beads comprise avidin. In embodiments, the chemically functionalized hydrogel particles comprise amine groups, carboxyl groups, hydroxyl groups, or a combination thereof. In embodiments, the hydrogel beads comprise a plurality of bifunctional monomers to functionalize the hydrogel beads with different chemicals and / or molecules.

[0181] In an embodiment, the hydrogel beads are functionalized to simulate one or more optical properties of target cells or labeled target cells. In an embodiment, the hydrogel beads include one or more high refractive index molecules. In an embodiment, the hydrogel beads include a variety of high refractive index molecules. In an embodiment, the high refractive index molecules enable the simulation of the SSC of target cells. In an embodiment, the high refractive index molecules are selected from one or more of the following: colloidal silica, alkyl acrylates, alkyl methacrylates, or a combination thereof. In an embodiment, the high refractive index molecules are alkyl acrylates, alkyl methacrylates, or both. In an embodiment, alkyl acrylates or alkyl methacrylates contain 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group. In an embodiment, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, 2-ethylhexyl, heptyl or octyl. In an embodiment, the alkyl group is branched. In an embodiment, the alkyl group is linear.

[0182] In embodiments, the hydrogel beads comprise a cell surface marker, an epitope binding region of a cell surface marker, or a combination thereof. V. Characteristics of Hydrogel Beads

[0183] In embodiments, the hydrogel beads described herein comprise artificial optical scattering properties that are substantially similar to corresponding optical scattering properties of target cells.

[0184] In embodiments, the target cell is a lymphocyte, a monocyte or a granulocyte. In embodiments, the target cell is a prokaryotic cell. In embodiments, the target cell is a eukaryotic cell. In embodiments, the target cell is a leukocyte. In embodiments, the target cell is a platelet. In embodiments, the target cell is an erythrocyte. In embodiments, the target cell is an immune cell. In embodiments, the immune cell is a T cell, a B cell, a NK cell, a lymphocyte, a monocyte, a granulocyte, a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage or a dendritic cell.

[0185] In embodiments, the optical scatter property is selected from side scatter (SSC), forward scatter (FSC), an angled light scatter profile, or a secondary marker profile (such as a fluorescent marker profile), an absorption profile, a fluorescence profile, or an emission profile.

[0186] In embodiments, artificial optical scattering properties are provided by: co-monomers, chemical side groups, encapsulated materials, colloidal silica, or the ratio of acrylamide to bisacrylamide.

[0187] In embodiments, the optical scattering property substantially similar to the corresponding optical scattering property of the target cell is SSC. In embodiments, the SSC of the hydrogel beads is within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the SSC of the target cell, as measured by a cell counting device. In embodiments, the side scattering of the hydrogel beads is tuned by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (about 100 nm) to the central aqueous phase prior to polymerization.

[0188] In an embodiment, the optical scattering property substantially similar to the corresponding optical scattering property of the target cell is FSC. In an embodiment, the forward scattering of the hydrogel beads can be adjusted by adjusting the refractive index of the hydrogel. In an embodiment, the refractive index of the hydrogel beads described herein can be measured using interferometry, dispersion method, Brewster Angle method or by using a refractometer. In an embodiment, the refractive index of the hydrogel can be adjusted by adding a comonomer. In an embodiment, the comonomer is allyl acrylate, allyl methacrylate or a combination thereof. Forward scattering can also be adjusted with side scattering nanoparticles containing sufficient optical resolution / size / density (including but not limited to a higher density colloidal suspension of silica and / or PMMA particles).

[0189] In embodiments, the hydrogel beads provided herein have a refractive index (RI) greater than about 1.10, greater than about 1.15, greater than about 1.20, greater than about 1.25, greater than about 1.30, greater than about 1.35, greater than about 1.40, greater than about 1.45, greater than about 1.50, greater than about 1.55, greater than about 1.60, greater than about 1.65, greater than about 1.70, greater than about 1.75, greater than about 1.80, greater than about 1.85, greater than about 1.90, greater than about 1.95, greater than about 2.00, greater than about 2.10, greater than about 2.20, greater than about 2.30, greater than about 2.40, greater than about 2.50, greater than about 2.60, greater than about 2.70, greater than about 2.80, or greater than about 2.90.

[0190] In embodiments, the refractive index (RI) of the hydrogel beads provided herein is from about 1.10 to about 3.0, or from about 1.15 to about 3.0, or from about 1.20 to about 3.0, or from about 1.25 to about 3.0, or from about 1.30 to about 3.0, or from about 1.35 to about 3.0, or from about 1.4 to about 3.0, or from about 1.45 to about 3.0, or from about 1.50 to about 3.0, or from about 1.6 to about 3.0, or from about 1.7 to about 3.0, or from about 1.8 to about 3.0, or from about 1.9 to about 3.0, or from about 2.0 to about 3.0.

[0191] In embodiments, the hydrogel beads provided herein have a refractive index (RI) of less than about 1.10, less than about 1.15, less than about 1.20, less than about 1.25, less than about 1.30, less than about 1.35, less than about 1.40, less than about 1.45, less than about 1.50, less than about 1.55, less than about 1.60, less than about 1.65, less than about 1.70, less than about 1.75, less than about 1.80, less than about 1.85, less than about 1.90, less than about 1.95, less than about 2.00, less than about 2.10, less than about 2.20, less than about 2.30, less than about 2.40, less than about 2.50, less than about 2.60, less than about 2.70, less than about 2.80, or less than about 2.90.

[0192] In an embodiment, the hydrogel beads have a refractive index greater than about 1.15. In an embodiment, the hydrogel beads have a refractive index greater than about 1.3. In an embodiment, the hydrogel beads have a refractive index greater than about 1.7.

[0193] In some embodiments, hydrogel particles of the present disclosure have material modulus properties (eg, elasticity) that more closely resemble those of target cells compared to polystyrene beads having the same diameter.

[0194] In embodiments, the size (e.g., diameter, width, thickness) of the hydrogel beads of the present disclosure is substantially similar to the target cells. In embodiments, the hydrogel beads have a diameter of less than about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 600 μm, about 800 μm, or less than about 1000 μm. In some embodiments, the hydrogel particles have a diameter greater than about 1 μm, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, 300, 350, 400, 450, 500, 600, 800, or greater than 1000 μm. In some embodiments, the diameter of the hydrogel particles is in the range of 2.5 μm to 100 μm. In some embodiments, the hydrogel particles have a diameter from about 2.5 μm to about 25 μm, from about 3 μm to about 20 μm, from about 3.5 μm to about 15 μm, from about 4 μm to about 12 μm, from about 5 μm to about 10 μm, from about 6 μm to about 9 μm, from about 7 μm to about 8 μm, or from about 10 μm to about 20 μm. In an embodiment, the hydrogel beads have a diameter of less than about 100 μm. In an embodiment, the hydrogel beads have a diameter of less than about 10 μm. In an embodiment, the hydrogel beads have a diameter greater than about 10 μm. In an embodiment, the hydrogel beads have a diameter from about 10 μm to about 20 μm. In an embodiment, the hydrogel beads have a diameter of less than about 1 μm. In an embodiment, the hydrogel beads have a diameter greater than about 1 μm. In an embodiment, the diameter of the hydrogel beads is measured using dynamic light scattering.

[0195] In embodiments, the hydrogel beads have a width of less than about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 600 μm, about 800 μm, or less than about 1000 μm. In some embodiments, the hydrogel particles have a width greater than about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 600 μm, about 800 μm, or about 1000 μm. In some embodiments, the width of the hydrogel particles is in the range of 2.5 μm to 100 μm. In some embodiments, the hydrogel particles have a width of from about 2.5 μm to about 25 μm, from about 3 μm to about 20 μm, from about 3.5 μm to about 15 μm, from about 4 μm to about 12 μm, from about 5 μm to about 10 μm, from about 6 μm to about 9 μm, or from about 7 μm to about 8 μm.

[0196] In embodiments, the hydrogel particles have a thickness of less than about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 600 μm, about 800 μm, or less than about 1000 μm. In some embodiments, the hydrogel particles have a thickness of greater than about 1 μm, about 2 μm, about 5 μm, about 10 μm, about 15 μm, about 20 μm, about 25 μm, about 30 μm, about 35 μm, about 40 μm, about 45 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 120 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 600 μm, about 800 μm, or less than about 1000 μm, including all ranges and subranges therebetween. In some embodiments, the thickness of the hydrogel particles is in the range of 2.5 μm to 100 μm. In some embodiments, the hydrogel particles have a thickness of from about 2.5 μm to about 25 μm, from about 3 μm to about 20 μm, from about 3.5 μm to about 15 μm, from about 4 μm to about 12 μm, from about 5 μm to about 10 μm, from about 6 μm to about 9 μm, or from about 7 μm to about 8 μm.

[0197] In embodiments, when labeled with a pro-apoptotic signal, the hydrogel beads exhibit a mean fluorescence intensity (MFI) that is at least as high as the MFI of target cells labeled with the same pro-apoptotic signal.

[0198] In embodiments, when labeled with a pro-apoptotic signal, the hydrogel beads exhibit a mean fluorescence intensity (MFI) that is substantially the same as the MFI of target cells labeled with the same pro-apoptotic signal.

[0199] In embodiments, the MFI of the hydrogel beads and the MFI of the target cells are within 10% to 50%. In embodiments, the MFI of the hydrogel beads and the MFI of the target cells are within 10%, within 11%, within 12%, within 13%, within 14%, within 15%, within 16%, within 17%, within 18%, within 19%, within 20%, within 21%, within 22%, within 23%, within 24%, within 25%, within 26%, within 27%, within 28%, within 29%, within 30%, within 31%, within 32%, within 33%, within 34%, within 35%, within 36%, within 37%, within 38%, within 39%, within 40%, within 41%, within 42%, within 43%, within 44%, within 45%, within 46%, within 47%, within 48%, within 49%, or within 50%, including all values ​​and subranges therebetween, inclusive.

[0200] In embodiments, the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%.

[0201] In embodiments, when labeled with a DNA binding dye, the hydrogel beads exhibit a mean fluorescence intensity (MFI) at least as high as the mean fluorescence intensity (MFI) of target cells labeled with the same DNA binding dye. In embodiments, when labeled with a DNA binding dye, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same DNA binding dye. VI. Kits containing hydrogel beads

[0202] In embodiments, a kit is provided comprising the hydrogel beads of Part IV described herein.

[0203] In an embodiment, the present invention provides a kit comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal binding agent of the first population of hydrogel beads; iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0204] In embodiments, the second hydrogel population and the third hydrogel population do not contain any nucleic acid. In embodiments, the second hydrogel population and the third hydrogel population do not contain any double-stranded DNA. In embodiments, the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a w / w ratio of about 1:1:1. In embodiments, the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a ratio of about 1:1:1 by number of beads.

[0205] In embodiments, the first, second, and third hydrogel bead populations are in the following w / w ratios: about 1:1:8, about 1:2:7, about 1:3:6, about 1:4:5, about 1:5:4, about 1:6:3, about 1:7:2, about 1:8:1, about 2:1:7, about 2:2:6, about 2:3:5, about 2:4:4, about 2:5:3, about 2:6:2, about 2:7:1 , about 3:1:6, about 3:2:5, about 3:3:4, about 3:4:3, about 3:5:2, about 3:6:1, about 4:1:5, about 4:2:4, about 4:3:3, about 4:4:2, about 4:5:1, about 5:1:4, about 5:2:3, about 5:3:2, about 5:4:1, about 6:1:3, about 6:2:2, about 6:3:1, about 7:1:2, about 7:2:1, or about 8:1:1. In embodiments,

[0206] In embodiments, the first, second, and third hydrogel bead populations are in the following ratios by number of beads: about 1:1:8, about 1:2:7, about 1:3:6, about 1:4:5, about 1:5:4, about 1:6:3, about 1:7:2, about 1:8:1, about 2:1:7, about 2:2:6, about 2:3:5, about 2:4:4, about 2:5:3, about 2:6:2, about 2:7:2 1, about 3:1:6, about 3:2:5, about 3:3:4, about 3:4:3, about 3:5:2, about 3:6:1, about 4:1:5, about 4:2:4, about 4:3:3, about 4:4:2, about 4:5:1, about 5:1:4, about 5:2:3, about 5:3:2, about 5:4:1, about 6:1:3, about 6:2:2, about 6:3:1, about 7:1:2, about 7:2:1, or about 8:1:1.

[0207] In embodiments, each of the first, second, and third hydrogel bead populations comprises about 10%-50% by weight of the total amount of hydrogel beads in the kit or composition. In embodiments, each of the first, second, and third hydrogel bead populations comprises about 10%-50% by weight of the total amount of hydrogel beads in the kit or composition. VII. Compositions Comprising Hydrogel Beads

[0208] In embodiments, provided are compositions comprising the hydrogel beads of Part IV described herein.

[0209] In an embodiment, the composition comprises: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal binding agent of the first population of hydrogel beads; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0210] In an embodiment, the composition comprises: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binding agent; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal binding agent of the first population of hydrogel beads; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0211] In an embodiment, the composition comprises: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, the third population of hydrogel beads comprising: i) polymerized monomers; but ii) lacking the pro-apoptotic signal of the first population of hydrogel beads; but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads.

[0212] In embodiments, the second hydrogel population and the third hydrogel population do not contain any nucleic acid. In embodiments, the second hydrogel population and the third hydrogel population do not contain any double-stranded DNA. In embodiments, the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a w / w ratio of about 1:1:1. In embodiments, the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a ratio of about 1:1:1 by the number of beads. In embodiments, each population in the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population accounts for about 10%-50% of the total amount of hydrogel beads in the kit or composition by weight. In embodiments, each population in the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population accounts for about 10%-50% of the total amount of hydrogel beads in the kit or composition by the number of beads.

[0213] In embodiments, the first, second, and third hydrogel bead populations are in the following w / w ratios: about 1:1:8, about 1:2:7, about 1:3:6, about 1:4:5, about 1:5:4, about 1:6:3, about 1:7:2, about 1:8:1, about 2:1:7, about 2:2:6, about 2:3:5, about 2:4:4, about 2:5:3, about 2:6:2, about 2:7:1 , about 3:1:6, about 3:2:5, about 3:3:4, about 3:4:3, about 3:5:2, about 3:6:1, about 4:1:5, about 4:2:4, about 4:3:3, about 4:4:2, about 4:5:1, about 5:1:4, about 5:2:3, about 5:3:2, about 5:4:1, about 6:1:3, about 6:2:2, about 6:3:1, about 7:1:2, about 7:2:1, or about 8:1:1. In embodiments,

[0214] In embodiments, the first, second, and third hydrogel bead populations are in the following ratios by number of beads: about 1:1:8, about 1:2:7, about 1:3:6, about 1:4:5, about 1:5:4, about 1:6:3, about 1:7:2, about 1:8:1, about 2:1:7, about 2:2:6, about 2:3:5, about 2:4:4, about 2:5:3, about 2:6:2, about 2:7:2 1, about 3:1:6, about 3:2:5, about 3:3:4, about 3:4:3, about 3:5:2, about 3:6:1, about 4:1:5, about 4:2:4, about 4:3:3, about 4:4:2, about 4:5:1, about 5:1:4, about 5:2:3, about 5:3:2, about 5:4:1, about 6:1:3, about 6:2:2, about 6:3:1, about 7:1:2, about 7:2:1, or about 8:1:1. VIII. Methods using hydrogel beads

[0215] In embodiments, methods for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells are provided herein. In embodiments, the method comprises: a) providing a hydrogel bead population as described herein or a kit or composition provided herein; b) contacting the hydrogel bead population with a pre-apoptotic signal and / or a DNA dye; c) measuring the concentration of a pre-apoptotic signal and / or a DNA dye in the hydrogel bead population; d) measuring the concentration of a pre-apoptotic signal and / or a DNA dye in the target cell sample; and e) comparing the concentration of a pre-apoptotic signal and / or a DNA dye measured in the hydrogel bead population and the target cell sample; thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0216] In an embodiment, provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population, kit or composition described herein, b) contacting the hydrogel bead population with a pre-apoptotic signal and / or a DNA dye; c) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; d) calibrating the cell counting device based on the measured concentration of the pre-apoptotic signal and / or DNA dye of the hydrogel beads; and e) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0217] In an embodiment, provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population described herein, or a hydrogel bead population from a kit or composition described herein; wherein at least a hydrogel bead population within the hydrogel bead population contains a pre-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the hydrogel bead population; c) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the target cell sample; and d) comparing the concentrations of the pre-apoptotic signal and / or DNA dye measured in the hydrogel bead population and the target cell sample; thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0218] In an embodiment, provided herein is a method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a hydrogel bead population described herein, or a hydrogel bead population from a kit or composition described herein; wherein at least a hydrogel bead population within the hydrogel bead population contains a pre-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; c) calibrating the cell counting device based on the measured concentration of the pre-apoptotic signal and / or DNA dye of the hydrogel beads; d) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0219] In embodiments, the hydrogel beads provided herein can be used to determine the dynamic range and / or sensitivity of the detection of specific cell surface markers or their combinations on target cell populations. In embodiments, the hydrogel beads can be tuned to have substantially the same side scatter and / or forward scatter as the target cells. In embodiments, the hydrogel particle sub-population is derived with a cell surface marker of a specific copy number. In embodiments, the cell surface marker is a cell surface receptor or its domain. In embodiments, the cell surface receptor or its domain is its epitope binding region. In embodiments, a separate hydrogel bead sub-population can be derived to have a cell surface marker with a unique copy number, for example, a sub-population will contain 100 copies of cell surface markers, a second sub-population will contain 1,000 copies of the same cell surface marker, and a third sub-population will contain 10,000 copies of the same cell surface. In embodiments, the hydrogel bead population is fluorescently stained for the corresponding cell surface markers, and the fluorescence of the hydrogel beads in each sub-population is detected. In this regard, the hydrogel particle sub-population can be used to generate a fluorescence emission standard curve for target cells with corresponding cell markers. The cell surface marker can be any cell surface marker or binding region thereof provided herein, or a cell surface marker known to a person of ordinary skill in the art. IX. Instruments

[0220] The hydrogel beads of the present disclosure can be used in a variety of cytometry applications. A non-limiting list of cytometry devices compatible with the hydrogels and methods disclosed herein is provided in Table 2 below. Table 2. Instruments used with the embodiments described herein Example

[0221] The following examples are given for the purpose of illustrating various embodiments of the present disclosure and are not intended to limit the present disclosure in any way. Those skilled in the art will recognize changes therein and other uses, which are within the spirit of the present disclosure defined by the scope of the claims. Example 1. Production of hydrogel beads

[0222] Photomasks for UV lithography were obtained from CADart Services Inc. and designed using AutoCad (AutoDesk, Inc.). SU-8 photoresist (Microchem, Inc.) was photocrosslinked onto a 4" silicon wafer using a collimated UV light source (OAI, Inc.) to create a master for microfluidic device fabrication. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using published standard methods for soft lithography and microfluidic device fabrication (see McDonald JC et al., 2000, Electrophoresis 21:27-40).

[0223] Droplets were formed using flow focusing geometry, where two oil channels focused the central flow of aqueous monomer solution to break the droplets in the water-in-oil emulsion. Fluorocarbon oil (Novec 7500 3M, Inc.) was used as the external continuous phase liquid for droplet formation. To stabilize the droplets prior to polymerization, a surfactant was added to the oil phase (ammonium carboxylate salt of Krytox 157 FSH, Dupont) at 0.5% w / w. To prepare basic polyacrylamide gel beads, a central phase of an aqueous monomer solution is used, which contains N-acrylamide (1%-20% w / v), a cross-linker that allows hydrogel cleavage (N,N'-bis(acryloyl)cystamine, bis(2-methacryloyl)oxyethyl disulfide, allyl disulfide, polyethylene glycol (PEG), N-hydroxysuccinimide (NHS) ester disulfide, acryloyl-PEG-disulfide-PEG-acryloyl, or 3-(2-pyridyldithio)propionic acid succinimidyl ester, diisopropylphenyl alcohol dimethacrylate, diisopropylphenyl alcohol diacrylate, 2,5-dimethyl-2,5-hexanediol dimethacrylate, acylhydrazone, or 3,9-divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane), an accelerator, and ammonium persulfate (1% w / v). An accelerator (N,N,N',N'tetramethylethylenediamine (2% vol%)) was added to the oil phase to initiate polymerization of the hydrogel beads after droplet formation.

[0224] Comonomers can be added to the base gel formulation to increase functionality. Allyl-amines provide primary amine groups for secondary labeling after gel formation. Forward scattering can be adjusted by adjusting the refractive index of the gel by adding comonomers allyl acrylate and allyl methacrylate. Side scattering of droplets can be tuned by adding a colloidal suspension of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles (about 100 nm) to the central aqueous phase before polymerization.

[0225] Stoichiometric multiplexing of hydrogel beads is achieved by utilizing comonomers containing chemically orthogonal side groups (amine, carboxyl, maleimide, epoxide, alkyne, etc.) for secondary labeling.

[0226] Droplets formed at an average rate of 5 kHz and were collected in the fluorocarbon oil phase.Polymerization was completed at 50°C in 30 minutes and the resulting hydrogel beads were washed from the oil into aqueous solution.

[0227] Three different hydrogel bead populations were prepared according to the above method. The first hydrogel bead population contained (i) polymerized monomers and bifunctional monomers, (ii) phosphatidylserine or anti-annexin V antibody or antigen-binding fragment thereof (pro-apoptotic signal binder), and (iii) encapsulated nucleic acid. The first hydrogel bead population can bind to both pro-apoptotic signals and DNA intercalating dyes, and thus act as a mimetic of dead cells.

[0228] The second hydrogel bead population contains (i) polymerized monomers and bifunctional monomers, and (ii) a pro-apoptotic signal binding agent (e.g., phosphatidylserine or an anti-Annexin V antibody or antigen-binding fragment thereof), but lacks the encapsulated nucleic acid of the first hydrogel bead population. The second hydrogel bead population can bind to the pro-apoptotic signal but cannot bind to the DNA intercalating dye, and thus acts as a mimetic of cells that are undergoing apoptosis but are not yet dead.

[0229] The third hydrogel bead population contains (i) polymerized monomers and bifunctional monomers, but lacks the pro-apoptotic signal binding agent and encapsulated nucleic acid of the first and second hydrogel bead populations. The third hydrogel bead population cannot bind to the pro-apoptotic signal or DNA intercalating dye, and thus acts as a mimetic of living cells.

[0230] A fourth hydrogel bead population is generated for live / dead assays. This fourth hydrogel bead population contains an amine-reactive dye binder and encapsulated nucleic acid, but does not contain the pro-apoptotic signal binder of the first hydrogel bead population. In the event that the signal of DNA or amine would be implicated in the dead population, this fourth bead population serves as a dual signal control for dead cells. Example 2. Hydrogel beads mimic forward and side scatter profiles of various cell types

[0231] This example describes the optional tuning of hydrogel beads to match the optical properties of one or more target cells. Different types of cells (e.g., granulocytes, monocytes, and lymphocytes) exhibit different optical scattering properties (e.g., forward scatter and side scatter). In some embodiments, the optical properties of hydrogel beads are tuned to mimic specific cell types. The tuning of the hydrogel can be performed via the methods described in U.S. Pat. No. 10,753,846. In brief, as Figure 74-Figure 76 As depicted in Figure 2, hydrogel beads can be tuned in multiple dimensions to match specific cell types. Using optical parameters such as FSC and SSC ( Fig.75 The hydrogel beads are further functionalized with stoichiometrically tuned ratios of specific chemical side groups and secondary markers, allowing the beads to be precisely matched to target cells without suffering from the biological noise associated with fixed cell lines. Fig.76 ). Figure 1It was shown that the hydrogel beads described herein can be tuned to match the forward scatter and side scatter of various cell populations. Example 3. Hydrogel beads can be dyed with both DNA intercalating dyes and amine reactive dyes

[0232] This example demonstrates the use of hydrogel beads of the present disclosure as a control for DNA and other amine binding dyes. The "fourth" hydrogel bead population of Example 1 was compared with a DNA intercalating dye ( Figure 2-Figure 5 ) and amine-reactive dyes ( Figure 6-Figure 9 ) are combined, the population containing (i) polymerized monomers and bifunctional monomers, (ii) amine dye binders and (iii) encapsulated nucleic acids. Figure 2-Figure 5 A fourth hydrogel bead population is shown with multiple DNA intercalating dyes. For example, the fourth hydrogel bead is with propidium iodide ("PI", Figure 2-Figure 3 ) and 7-aminoactinomycin D ("7AAD", Figure 4-Figure 5 )combined. Figure 6-Figure 9 The fourth hydrogel beads are shown with the amine reactive dye GHOST DYE TM ( Figure 6-Figure 7 ) and LIVE / DEAD TM Blue (fixable Figure 8-Figure 9 )combined. Figure 10-12 The fourth hydrogel beads are shown to be simultaneously coated with a DNA intercalating dye (e.g., 7AAD) and an amine reactive dye (e.g., GHOSTDYE TM )combined.

[0233] The ability of the hydrogel beads to bind both amine-reactive dyes and DNA intercalating dyes allows the beads to serve as a dead cell control, depending on the requirements of the assay. Example 4. As a viability control, hydrogel beads outperform heat-killed cells and polystyrene beads

[0234] Viability controls are used in flow cytometry to distinguish live cells, dead cells, and apoptotic cells. The following were compared for their ability to act as viability controls: (i) the hydrogel beads of Example 1 (comprising the fourth, second, and third hydrogel bead populations); (ii) a mixture of heat-killed lymphocytes and live lymphocytes, and (iii) amine-reactive polystyrene beads. Each of (i), (ii), and (iii) was treated with the DNA intercalating dye 7AAD and the amine-reactive dye GHOST DYE. TM The DNA intercalating dye and GHOST DYE in each sample were evaluated on a cell counting device. TM concentration.

[0235] Figure 13-Figure 15 The following forward scatter and side scatter are shown: (i) Hydrogel beads of Example 1 ( Fig.14, labeled as “hydrogel beads”), (ii) a mixture of heat-killed lymphocytes and live lymphocytes ( Fig.13 ), and (iii) amine-reactive polystyrene beads ( Fig.15 (i) The hydrogel beads of Example 1 exhibited substantially the same side scatter as lymphocytes. Fig.14 and Fig.13 In contrast, (iii) the side scatter of amine-reactive polystyrene beads differs from that of lymphocytes ( Fig.15 and Fig.13 for comparison).

[0236] Figure 16-Figure 17 The DNA intercalating dye 7AAD is shown to bind to: (i) the hydrogel beads of Example 1 ( Fig.17 ) and (ii) a mixture of heat-killed lymphocytes and live lymphocytes ( Fig.16 ). However, (iii) amine-reactive polystyrene beads did not bind to 7AAD.

[0237] Figure 18-Figure 20 Shown is the amine-reactive dye GHOST DYE TM Combined with: (i) the hydrogel beads of Example 1 ( Fig.19 ), (ii) a mixture of heat-killed lymphocytes and live lymphocytes ( Fig.18 ), and (iii) amine-reactive polystyrene beads ( Fig. 20 ).

[0238] Unlike amine-reactive polystyrene beads that bind only to amine-reactive dyes, the fourth hydrogel bead population of Example 1 binds to both DNA intercalating dyes and amine-reactive dyes. Therefore, as a viability control, the hydrogel beads of Example 1 outperform polystyrene beads and provide scientists with versatility and multiple options.

[0239] As a viability control, the hydrogel beads of Example 1 also outperformed a mixture of heat-killed lymphocytes and live lymphocytes. The generation of heat-killed lymphocyte and live lymphocyte controls is time-consuming because it requires heat killing of a portion of the lymphocytes. In addition, dead lymphocytes emit higher autofluorescence and experience higher levels of nonspecific binding compared to live cells. The use of hydrogel beads alleviated these problems, thereby improving consistency between experiments. Example 5. The hydrogel beads of Example 1 simulate a target cell population containing living cells, dead cells, and cells undergoing apoptosis

[0240] Compositions containing the first, second, and third hydrogel bead populations of Example 1 allow identification of live cells, dead cells, and cells undergoing apoptosis. The first hydrogel bead population contains both a pro-apoptotic signal binding agent (e.g., phosphatidylserine ("PS")) and an encapsulated nucleic acid. The second hydrogel bead population contains a pro-apoptotic signal binding agent, but does not contain an encapsulated nucleic acid. The third hydrogel bead population contains neither a pro-apoptotic signal binding agent nor an encapsulated nucleic acid.

[0241] The first, second and third hydrogel bead populations were stained with annexin V, which was labeled with fluorescein isothiocyanate (FITC) dye. The annexin V was bound to PS. The hydrogel beads were also stained with 7AAD, a DNA intercalating dye, which intercalates between DNA bases and exhibits fluorescence. These three hydrogel populations were subsequently evaluated on a flow cytometer device.

[0242] Fig.21 The binding of three hydrogel bead populations to Annexin V and 7AAD is shown. The third hydrogel bead population binds neither Annexin V nor 7AAD and is labeled "live cell mimic". The second hydrogel bead population binds to Annexin V, but not 7AAD, and is labeled "apoptotic cell mimic". The first hydrogel bead population binds to both Annexin V and 7AAD and is labeled "dead cell mimic".

[0243] The use of the first, second, and third hydrogel bead populations provides an improvement over the live / dead assay of Example 4 (which uses the second, third, and fourth hydrogel bead populations). The hydrogel beads in Example 4 are able to distinguish dead cells (beads containing an amine dye binder and encapsulated nucleic acid) from live cells (lacking the amine dye binder and encapsulated nucleic acid of the fourth population).

[0244] Advantageously, the first, second and third hydrogel colonies tested in this embodiment serve as the analogs of the cell colonies containing living cells, dead cells and cells undergoing apoptosis. Living cells do not contain DNA accessible to DNA intercalating dyes, and do not have exposed PS apoptosis signal binders. Therefore, the third hydrogel bead colony that is neither bound to annexin V nor to 7AAD serves as a living cell analog. Cells undergoing apoptosis have exposed phosphatidylserine, and therefore can be combined with annexin V. Therefore, the second hydrogel bead colony that is bound to annexin V but not to 7AAD serves as a analog of cells undergoing apoptosis. Dead cells have ruptured cell membranes, and therefore have exposed PS and DNA. Therefore, the first hydrogel bead colony that is bound to both annexin V and DNA serves as a analog of dead cells. Example 6. Hydrogel beads are stable for at least 37 days

[0245] Stability of the Hydrogel Beads of the Present Disclosure Compositions were prepared containing equal amounts of three different hydrogel bead populations.

[0246] The first hydrogel bead population contains (i) polymerized monomers and bifunctional monomers, (ii) anti-Annexin V antibodies or antigen-binding fragments thereof (pro-apoptotic signal binders), and (iii) encapsulated nucleic acids. The first hydrogel bead population can bind to both pro-apoptotic signals and DNA intercalating dyes and thus act as a mimetic of dead cells.

[0247] The second hydrogel bead population contains (i) polymerized monomers and bifunctional monomers, and (ii) a pro-apoptotic signal binding agent (e.g., an anti-Annexin V antibody or an antigen-binding fragment thereof), but lacks the encapsulated nucleic acid of the first hydrogel bead population. The second hydrogel bead population can bind to the pro-apoptotic signal but cannot bind to the DNA intercalating dye, and thus acts as a mimetic of cells that are undergoing apoptosis but are not yet dead.

[0248] The third hydrogel bead population contains (i) polymerized monomers and bifunctional monomers, but lacks the pro-apoptotic signal binding agent and encapsulated nucleic acid of the first hydrogel bead population. The third hydrogel bead population cannot bind to the pro-apoptotic signal or DNA intercalating dye, and thus acts as a mimetic of living cells.

[0249] The composition was stored at 4°C for 37 days. Before storage ("Day 0") and one, seven, fourteen and thirty-seven days after storage, the composition was stained with an annexin V dye (labeled with FITC dye) conjugated to an anti-annexin V antibody and a DNA intercalating dye 7AAD. The composition was then evaluated on a flow cytometer apparatus.

[0250] Figure 22-Figure 26 Shown on day 0 ( Fig. 22 ); one day after storage ( Fig.23 ); seven days( Fig.24 ); Fourteen days ( Fig.25 ); and thirty-seven days ( Fig.26 ), binding of the composition to Annexin V and 7AAD. During 37 days of storage at 4°C, the third hydrogel bead population bound neither to Annexin V nor to 7AAD, demonstrating stability and avoidance of nonspecific binding. During 37 days of storage at 4°C, the second hydrogel bead population bound to Annexin V but not to 7AAD. During 37 days of storage at 4°C, the first hydrogel beads bound to both Annexin V and 7AAD.

[0251] This experiment showed that when the hydrogel beads were stored at 4°C, the hydrogel bead population remained stable and could therefore serve as a mimic of live, dead, and apoptotic cells for at least 37 days. Example 7. As a compensatory control, a population of hydrogel beads stained with a single dye outperforms live cells stained with a single dye

[0252] The following compositions (each stained with a single dye) were evaluated for their ability to serve as compensation controls: (a) compositions containing the first, second, and third hydrogel bead populations of Example 1; (b) Jurkat cells; and (c) peripheral blood mononuclear cells (PBMCs).

[0253] A composition containing three hydrogel bead populations was prepared by combining the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population in equal ratios.

[0254] Each of the following samples was analyzed by ALEXA 647 dye-labeled annexin V or 7AAD staining: (a) Compositions containing three hydrogel bead populations, (b) PBMCs, and (c) Jurkat cells. Aurora cell counting device or BD FACSLyric TM The composition was evaluated above.

[0255] Figure 27-Figure 31 Shown in BD FACSLyric TM Cell counting device ( Figure 27-Figure 29 )or Aurora Cell Counting Device ( Figure 30-Figure 31 ) and 7AAD binding to the following samples stained with 7AAD alone: ​​(a) a composition containing three hydrogel bead populations ( Fig. 27 , Fig.30 ), (b) Jurkat cells ( Fig.28 , Fig.31 ), and (c) PBMCs ( Fig.29 ).

[0256] Figure 32-Figure 36 Shown in BD FACSLyric TM Cell counting device ( Figure 32-Figure 34 )or Aurora Cell Counting Device ( Figure 35-Figure 36 ) and the binding of Annexin V to the following samples stained with Annexin V alone: ​​(a) a composition containing three hydrogel bead populations ( Fig.32 , Fig.35 ), (b) Jurkat cells ( Fig.33 , Fig.36 ), and (c) PBMCs ( Fig.34 ).

[0257] When the hydrogel bead composition was stained with a single dye (7AAD or Annexin V), the flow cytometry dot plots showed clear positive (populations bound to 7AAD or Annexin V) and negative (populations not bound to 7AAD or Annexin V) populations. In contrast, the dot plots of cells stained with a single dye did not show clear positive or negative populations. Fig.30 and Fig.31 This trend was independent of the cell counting device. This suggests that the hydrogel bead composition stained with a single dye is superior to the control of cells stained with a single dye. Example 8. Hydrogel bead populations stained with a single dye outperform live cells stained with a single dye as gating controls, compensation controls, and for spectral unmixing

[0258] The following compositions were evaluated for their ability to serve as gating controls, compensation controls, and tools for spectral unmixing: (a) compositions containing the first, second, and third hydrogel bead populations of Example 1; (b) Jurkat cells; and (c) peripheral blood mononuclear cells (PBMCs).

[0259] A composition containing three hydrogel bead populations was prepared by combining the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population in equal ratios.

[0260] Composition (a) was aliquoted into 96-well plates (100 μL of hydrogel beads suspended in annexin V buffer per well) and stained with 7AAD and / or with ALEXA Annexin V was stained with 647 dye.

[0261] Composition (b) was prepared by suspending Jurkat cells at a concentration of 10 million cells / mL in Annexin V buffer. Composition (b) was aliquoted into 96-well plates (100 μL of composition (b) per well). Composition (b) was treated with CD95 (0.0625 μg / mL) at 37°C for one hour and then washed three times with Annexin V buffer by centrifugation at 400×g for five minutes. Composition (b) was then treated with 7AAD and / or with ALEXA Annexin V 647 dye-labeled staining was performed for thirty minutes.

[0262] Composition (c) was prepared by suspending previously cryopreserved PBMCs at a concentration of 5 million cells / mL in annexin V buffer. Composition (c) was aliquoted into 96-well plates (100 μL of composition (c) per well). Composition (c) was treated with CD95 (0.0625 μg / mL) at 37°C for one hour, and then washed three times with annexin V buffer by centrifugation at 400×g for five minutes. Composition (c) was then treated with 7AAD and / or with ALEXA Annexin V 647 dye-labeled staining was performed for thirty minutes.

[0263] After staining, each of compositions (a), (b) and (c) was washed three times with Annexin V buffer by centrifugation at 400 xg for five minutes.

[0264] Resuspend the sample in Annexin V buffer. Aurora cell counting device or BD FACSLyric TM 500,000 cells from each sample were evaluated on a cell counting device. BD FACSLyric TM Fluorescence spillover on cell counting devices. Fluorescence spectra of the combinations (a), (b) and (c) labeled with 647 dye-labeled annexin V Spectral unmixing was performed on the Aurora cell counting device.

[0265] Figure 37-Figure 39 As shown in the BD FACSLyric TM The following forward scatter ("FSC") and side scatter ("SSC") were measured on a cell counting instrument: (a) a composition containing three hydrogel bead populations ( Fig.37 ); (b) Jurkat cells ( Fig.38 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.39 ). Figure 40-Figure 42 As shown in the BD FACSLyric TM The following forward scatter and side scatter were measured on a cell counting device: (a) a composition containing three hydrogel bead populations ( Fig.40 ); (b) Jurkat cells ( Fig.41 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.42 Each composition was gated to exclude debris (see Figure 37-Figure 42 circle on top).

[0266] The gated cells and / or beads are analyzed on each cytometry device to determine which beads or cells bind to 7AAD and / or Annexin V. BD FACSLyric TM Results on the cell counting device

[0267] Cells or beads from each of compositions (a), (b) and (c) are bound to 7AAD and / or stained with ALEXA Annexin V labeled with 647 dye, as in BD FACSLyric TM Compared with composition (b) Jurkat cells or composition (c) PBMC, composition (a) comprising three hydrogel bead populations and BD FACS Lyric TM The automatic compensation algorithm on the cell counting device is more compatible. Additionally, the distinction between the positive and negative populations is clearer in composition (a) than in the two cell populations (b) and (c).

[0268] Figure 43-Figure 45 is a histogram showing the TM The mean fluorescence intensity of 7AAD in the following: (a) a composition containing three hydrogel bead populations ( Fig.43 ); (b) Jurkat cells ( Fig.44 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.45 ).

[0269] Figure 52-Figure 54 is a histogram showing the TM The following measurements were made using ALEXA on the cell counting device: Average fluorescence intensity of 647 dye-labeled annexin V: (a) Compositions containing three hydrogel bead populations ( Fig.52 ); (b) Jurkat cells ( Fig.53 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.54 ).

[0270] Figure 61-Figure 63 As shown in the BD FACSLyric TM 7AAD measured on a cell counting device and with ALEXA Binding of 647 dye-labeled annexin V to: (a) a composition containing three hydrogel bead populations ( Fig.61 ); (b) Jurkat cells ( Fig.62 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.63). Live cells, dead cells, and apoptotic cells (labeled as "pre-apoptotic") are labeled in each figure. BD FACSLyric TM Fluorescence spillover on the cell counting device. Results on the Aurora Cell Counting Device

[0271] Cells or beads from each of compositions (a), (b) and (c) are bound to 7AAD and / or stained with ALEXA 647 dye-labeled annexin V, as in Detected on the Aurora cell counting device.

[0272] Figure 46-Figure 51 is a histogram that shows that Mean fluorescence intensity of 7AAD measured on the Aurora cytometer in: (a) a composition containing three hydrogel bead populations ( Fig.46 , Fig.49 ); (b) Jurkat cells ( Fig.47 , Fig.50 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.48 , Fig.51 ).exist Figure 46-Figure 48 In the use of combined Spectral unmixing of the fluorescence spectra of Jurkat cells labeled with 647 dye Annexin V. Figure 49-Figure 51 In the use of combined Fluorescence spectra of PBMCs labeled with 647 dye Annexin V were spectrally unmixed.

[0273] Figure 55-Figure 60 is a histogram that shows that The following measurements were made using the Aurora cell counting device: Average fluorescence intensity of 647 dye-labeled annexin V: (a) Compositions containing three hydrogel bead populations ( Fig.55 , Fig.58 ); (b) Jurkat cells ( Fig.56 , Fig.59 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.57 , Fig.60 ).exist Figure 55-Figure 57 In the use of combined Spectral unmixing of the fluorescence spectra of Jurkat cells labeled with 647 dye Annexin V. Figure 58-Figure 60 In the ALEXA Fluorescence spectra of PBMCs labeled with 647 dye Annexin V were spectrally unmixed.

[0274] Figure 64-Figure 72 As shown in 7AAD measured on the Aurora cell counting device and with ALEXA Binding of 647 dye-labeled annexin V to: (a) a composition containing three hydrogel bead populations ( Fig.64 , Fig.67 , Fig.70 ); (b) Jurkat cells ( Fig.65 , Fig.68 , Fig.71 ); or (c) peripheral blood mononuclear cells (PBMC) ( Fig.66 , Fig.69 , Fig.72 In each figure, live cells, dead cells, and apoptotic cells (labeled "pre-apoptotic") are labeled. Compositions containing three hydrogel bead populations of 647 dye-labeled annexin V and / or 7AAD ( Figure 70-Figure 72 ), Jurkat cells ( Figure 64-Figure 66 ) or PBMC( Figure 67-Figure 69 )’s fluorescence spectra were spectrally unmixed. in conclusion

[0275] This data shows that the hydrogel bead compositions can be used as gating controls, as compensation controls, and as a tool for spectral unmixing.

[0276] The hydrogel bead compositions are superior to cell controls because they are expressed using pro-apoptotic signals and viability dyes (e.g., using ALEXA The hydrogel bead compositions stained with 647 dye-labeled annexin V or 7AAD exhibited clear positive and negative bead populations. In contrast, the separation between positive and negative cell populations was less clear ( Fig.67 and Fig.69 for comparison).

[0277] The hydrogel bead composition is also superior to the cell control because the number of beads that act as mimics of live cell controls (i.e., beads from the third hydrogel bead population), dead cell controls (i.e., beads from the first hydrogel bead population), and apoptotic cell controls (i.e., beads from the second hydrogel bead population) can be adjusted. In contrast, the amount of dead cells, live cells, and apoptotic cells that act as apoptotic controls in a cell population cannot be precisely controlled. Therefore, a hydrogel bead composition can be produced in which beads that act as mimics of dead cells, live cells, and apoptotic cells each account for 33%.

[0278] The hydrogel bead compositions also exhibit less variability than cells. Different batches of cells can exhibit different properties depending on the age of the cells. For example, the forward scatter and side scatter of Jurkat cells of different ages are different. Fig.38 and Fig.73 For comparison, Fig.38 The forward and side scatter of one-day-old Jurkat cells are shown. Fig.73 The forward and side scatter of five-day-old Jurkat cells are shown. In contrast, three replicates of the hydrogel bead population showed consistent forward scatter, as well as consistent scatter with 7AAD and with ALEXA. Mean fluorescence intensity of 647 dye-labeled annexin V binding. Fig.37 , Fig.40 , Fig.43 , Fig.46 , Fig.49 , Fig.52 , Fig.55 , Fig.58 , Fig.61 , Fig.64 and Fig.67 Overlay these repetitions.

[0279] Additionally, using the hydrogel bead composition as a control is less time consuming than using a cell population as a control. The hydrogel bead composition does not require cell culture, does not require Annexin V buffer, and does not require induction of apoptosis. In contrast, the hydrogel bead composition is stable for 37 days at 4°C and can be immediately stained with a viability dye. Numbered Embodiments of the Disclosure

[0280] Notwithstanding the appended claims, the present disclosure sets forth the following numbered embodiments: 1. A hydrogel bead comprising: a) polymerizable monomers and difunctional monomers; and b) Pro-apoptotic signal binders. 2. A hydrogel bead, comprising: a) polymerizable monomers and difunctional monomers; and b) Pro-apoptotic signals. 3. A hydrogel bead according to embodiment 1 or 2, wherein the pro-apoptotic signal comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of the following: annexin V (UniProt Accession No. P08758), a polypeptide of SEQ ID NO: 103, an apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (UniProt Accession No. D9IWP9) (e.g., domain V), prothrombin or a fragment thereof (UniProt Accession No. P00734), milk fat globule-EGF-factor 8 (MFG-E8) (UniProt Accession No. Q08431), a phosphatidylserine receptor or a fragment thereof, SEQ ID NO: 102, CD36 (UniProt Accession No. P16671), LDL receptor-associated protein (UniProt Accession No. P01130), or an anti-calreticulin antibody or an antigen-binding fragment thereof. 4. The hydrogel beads of embodiment 3, wherein the pro-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof. 5. The hydrogel bead according to embodiment 4, wherein the phosphatidylserine receptor or fragment thereof comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of the following: brain-specific angiogenesis inhibitor-1 (Bai1) (UniProt Accession No. O41514), Axl (UniProt Accession No. P30530), Tyro3 (UniProt Accession No. Q06418), Mer (UniProt Accession No. Q12866 or UniProt Accession No. Q50744), TIM-1 (also known as "KIM-1") (UniProt Accession No. Q96D42), TIM-4 (UniProt Accession No. Q96H15), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (UniProt Accession No. P78380), stabilizer-1 (UniProt accession number Q9NY15), stabilizer-2 (UniProt accession number Q8WWQ8), CD300a (UniProt accession number Q9UGN4), CD300b (UniProt accession number A8K4G0), CD300f (UniProt accession number Q8TDQ1), receptor for advanced glycation end products (RAGE) (UniProt accession number Q15109), complement component 1q (C1q) (UniProt accession number P02746; UniProt accession number P02745; or UniProt accession number P02747), β2-glycoprotein I (β2GPI) and integrin αVβ3 / β5 (UniProt accession number P06756; UniProt accession number P05106; or UniProt accession number P18084). 6. A hydrogel bead according to embodiment 1, wherein the pro-apoptotic signal binder comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity with any one of the following: phosphatidylserine, anti-annexin V antibody or its antigen-binding fragment, annexin I (UniProt Accession No. P04083), calreticulin (UniProt Accession No. Q96L12), anti-CD36 antibody or its antigen-binding fragment, thrombospondin-1 (TSP-1) (UniProt Accession No. P07996), anti-β2-glycoprotein I antibody or its antigen-binding fragment, anti-milk fat globule-EGF-factor 8 (MFG-E8) or its antigen-binding fragment, anti-phosphatidylserine receptor or its antigen-binding fragment and anti-LDL receptor-related protein or its antigen-binding fragment. 7. The hydrogel bead according to any one of embodiments 1-6, comprising: c) Encapsulated nucleic acid. 8. The hydrogel bead of embodiment 7, wherein the nucleic acid is double-stranded DNA. 9. The hydrogel bead of any one of embodiments 2-3, 7 and 8, wherein the pro-apoptotic signal is annexin V. 10. The hydrogel bead of any one of embodiments 1, 3, 7, and 8, wherein the pro-apoptotic signal binding agent is phosphatidylserine. 11. The hydrogel bead of any one of embodiments 1, 3, 7 and 8, wherein the pro-apoptotic signal binding agent is an anti-Annexin V antibody or an antigen-binding fragment thereof. 12. The hydrogel bead of any one of embodiments 2-3, 7 and 8, wherein the pro-apoptotic signal is apo-15. 13. A hydrogel bead according to any one of embodiments 1-12, wherein the hydrogel bead comprises artificial optical scattering properties that are substantially similar to corresponding optical scattering properties of target cells, wherein the artificial optical scattering properties are provided by: comonomers, chemical side groups, encapsulated materials, colloidal silica, or a ratio of acrylamide to bisacrylamide. 14. The hydrogel bead of any one of embodiments 1-13, wherein the hydrogel bead comprises a scattering modulating additive. 15. The hydrogel bead of embodiment 13, wherein the optical scattering property substantially similar to the corresponding optical scattering property of the target cell is side scatter (SSC). 16. The hydrogel bead of embodiment 13, wherein the optical scattering property substantially similar to the corresponding optical scattering property of the target cell is forward scatter (FSC). 17. The hydrogel bead of embodiment 14, wherein the scattering modulating additive comprises a comonomer. 18. The hydrogel bead of embodiment 14, wherein the scattering modulating additive comprises a suspension of nanoparticles. 19. The hydrogel bead of any one of embodiments 13 and 15-18, wherein the target cell is one of a lymphocyte, a monocyte, or a granulocyte. 20. The hydrogel bead of any one of embodiments 13 and 15-18, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell. 21. The hydrogel bead of any one of embodiments 13 and 15-18, wherein the target cells are leukocytes. 22. The hydrogel bead of any one of embodiments 13 and 15-18, wherein the target cell is an immune cell. 23. The hydrogel bead of any one of embodiments 1-22, wherein the polymerized monomer is a biodegradable monomer. 24. The hydrogel bead of any one of embodiments 1-22, wherein the hydrogel bead is biodegradable. 25. The hydrogel bead of embodiment 23, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 26. The hydrogel bead of embodiment 23, wherein the hydrogel bead comprises a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 27. The hydrogel bead of embodiment 23, wherein the biodegradable monomer is a structural polysaccharide. 28. The hydrogel bead of embodiment 23, wherein the hydrogel bead comprises a structural polysaccharide. 29. The hydrogel beads according to embodiment 23, wherein the biodegradable monomer is selected from agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan polysaccharide, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp polysaccharide, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, Gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan and combinations thereof. 30. The hydrogel beads of embodiment 23, wherein the hydrogel beads comprise agar, agarose, alginic acid, polysaccharide acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp laminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof. 31. A hydrogel bead according to any one of embodiments 9 and 13-30, wherein the annexin V is from a species selected from the group consisting of: human, rabbit, mouse, giant panda, Peruvian night monkey, minke whale, blue whale, zebu x Bos indicus x Bos taurus, zebu, yak, cattle, buffalo, common marmoset, Bactrian camel, Chinese native dog, goat, Philippine tarsier, American beaver, cattle, Panamanian capuchin, Canadian elk, red deer, Tarim Hara elk, beluga whale, Osso's kangaroo rat, desert flag-tailed kangaroo rat, Indian elephant, Przewalski's horse, gray whale, domestic cat, western gorilla, eastern gorilla, wolverine, gray seal, striped hyena, silvery gibbon, multi-striped ground squirrel, African jerboa, Pacific short-beaked dolphin, ring-tailed lemur, white-fin dolphin, African savanna elephant, long-tailed macaque, macaque , drill, yellow-bellied marmot, Alpine marmot, American marmot, forest musk deer, barking deer, pet mink, American ferret, Hawaiian monk seal, white-cheeked gibbon, raccoon dog, Pacific walrus, white-tailed deer Texas subspecies, killer whale, sheep, chimpanzee, East African baboon, Pacific pocket mouse, harbor seal, sperm whale, Ugandan red colobus monkey, Cluys's crested lemur, Svalbard reindeer, Yunnan golden monkey, Amazon squirrel monkey, East American squirrel, shrew, Eurasian wild boar, black leaf monkey, tree shrew, bottlenose dolphin, Arctic ground squirrel, polar bear, Arctic fox and California sea lion. 32. A hydrogel bead according to any one of embodiments 13 and 13-31, wherein the annexin V comprises a sequence that exhibits at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity to a sequence selected from SEQ ID NOs: 1-101. 33. The hydrogel bead of any one of embodiments 7-32, wherein the encapsulated nucleic acid is bound to a dye. 34. The hydrogel beads according to embodiment 33, wherein the dye bound to the encapsulated nucleic acid is selected from 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), DRAQ5 TM 、DRAQ7 TM 、CytoPhase TM Violet, Helix NP TM Blue, Helix NP TM Green, Helix NP TM NIR、YOYO TM -1.TOTO TM -1 iodide (Thermo Fisher Scientific), SYTOX TM Blue, ethidium bromide, SYBR TM Gold, SYBR TM Green, SYBR TM Safe, and crystal violet. 35. The hydrogel bead of embodiment 34, wherein the dye is 7AAD. 36. The hydrogel bead of embodiment 34, wherein the dye is propidium iodide. 37. The hydrogel bead of any one of embodiments 1-36, wherein the hydrogel bead has a refractive index greater than about 1.15. 38. The hydrogel bead of any one of embodiments 1-36, wherein the hydrogel bead has a refractive index greater than about 1.3. 39. The hydrogel bead of any one of embodiments 1-36, wherein the hydrogel bead has a refractive index greater than about 1.7. 40. The hydrogel bead of any one of embodiments 1-39, wherein the hydrogel bead has a diameter of less than about 100 μm. 41. The hydrogel bead of any one of embodiments 1-39, wherein the hydrogel bead has a diameter greater than about 10 μm. 42. The hydrogel bead of any one of embodiments 1-39, wherein the hydrogel bead has a diameter greater than about 1 μm. 43. A hydrogel bead according to any one of embodiments 14-42, wherein the scattering modulation additive comprises polymer nanoparticles. 44. The hydrogel beads of embodiment 43, wherein the polymer nanoparticles comprise polystyrene. 45. The hydrogel bead of any one of embodiments 1-44, wherein the hydrogel bead is a chemically functionalized hydrogel particle. 46. ​​The hydrogel bead of any one of embodiments 1-45, wherein the hydrogel bead comprises free amine groups. 47. A hydrogel bead according to embodiment 46, wherein the pro-apoptotic signal binder is attached to the free amine group. 48. The hydrogel bead of any one of embodiments 1-47, wherein the hydrogel bead comprises allylamine. 49. A hydrogel bead according to any one of embodiments 2-5, 7-9 and 12-48, wherein when labeled with the pro-apoptotic signal, the hydrogel bead exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal. 50. A hydrogel bead according to any one of embodiments 2-5, 7-9 and 12-48, wherein when labeled with the pro-apoptotic signal, the hydrogel bead exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal. 51. A hydrogel bead according to any one of embodiments 1, 6-8, 10-11 and 13-48, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel bead exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal binding agent. 52. A hydrogel bead according to any one of embodiments 1, 6-8, 10-11 and 13-48, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel bead exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal binding agent. 53. The hydrogel bead of embodiment 50 or 52, wherein the MFI of the hydrogel bead and the MFI of the target cell are within 50%, 40%, 30%, 20% or 10%. 54. The hydrogel bead of any one of embodiments 7-53, wherein when labeled with a DNA binding dye, the hydrogel bead exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same DNA binding dye. 55. The hydrogel bead of any one of embodiments 7-53, wherein when labeled with a DNA binding dye, the hydrogel bead exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same DNA binding dye. 56. The hydrogel bead of embodiment 55, wherein the MFI of the hydrogel bead and the MFI of the target cell are within 50%, 40%, 30%, 20% or 10%. 57. A kit, comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) a pro-apoptotic signal binder; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lacking a pro-apoptotic signal binding agent in said first population of hydrogel beads; and iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 58. A composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) a pro-apoptotic signal binder; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lacking a pro-apoptotic signal binding agent in said first population of hydrogel beads; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 59. A kit comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lack of pro-apoptotic signals of said first population of hydrogel beads; and iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 60. A composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lack of pro-apoptotic signals of said first hydrogel bead population; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 61. The kit or composition of any one of embodiments 57-60, wherein the second hydrogel population and the third hydrogel population do not contain any nucleic acid. 62. The kit or composition of any one of embodiments 57-60, wherein the second hydrogel population and the third hydrogel population do not contain any double-stranded DNA. 63. The kit or composition of any one of embodiments 57-62, wherein the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a w / w ratio of about 1:1:1. 64. The kit or composition of any one of embodiments 57-62, wherein the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a ratio of about 1:1:1 by number of beads. 65. The kit or composition of any one of embodiments 57-62, wherein each of the first, second, and third hydrogel bead populations comprises approximately 10%-50% by weight of the total amount of hydrogel beads in the kit or composition. 66. The kit or composition of any one of embodiments 57-62, wherein each of the first, second, and third hydrogel bead populations comprises approximately 10%-50% of the total amount of hydrogel beads in the kit or composition, by number of beads. 67. A kit or composition according to any one of embodiments 59-66, wherein the pro-apoptotic signal comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any of the following: annexin V (UniProt Accession No. P08758), a polypeptide of SEQ ID NO: 103, an apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (UniProt Accession No. D9IWP9) (e.g., domain V), prothrombin or a fragment thereof (UniProt Accession No. P00734), milk fat globule-EGF-factor 8 (MFG-E8) (UniProt Accession No. Q08431), a phosphatidylserine receptor or a fragment thereof, SEQ ID NO: 102, CD36 (UniProt Accession No. P16671), LDL receptor-associated protein (UniProt Accession No. P01130), or an anti-calreticulin antibody or an antigen-binding fragment thereof. 68. A kit or composition according to any one of embodiments 59-66, wherein the pro-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof. 69. The kit or composition of embodiment 68, wherein the phosphatidylserine receptor or fragment thereof comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of: brain-specific angiogenesis inhibitor-1 (Bai1) (UniProt Accession No. O41514), Axl (UniProt Accession No. P30530), Tyro3 (UniProt Accession No. Q06418), Mer (UniProt Accession No. Q12866 or UniProt Accession No. Q50744), TIM-1 (also known as "KIM-1") (UniProt Accession No. Q96D42), TIM-4 (UniProt Accession No. Q96H15), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (UniProt Accession No. Accession No. P78380), stabilizer-1 (UniProt Accession No. Q9NY15), stabilizer-2 (UniProt Accession No. Q8WWQ8), CD300a (UniProt Accession No. Q9UGN4), CD300b (UniProt Accession No. A8K4G0), CD300f (UniProt Accession No. Q8TDQ1), receptor for advanced glycation end products (RAGE) (UniProt Accession No. Q15109), complement component 1q (C1q) (UniProt Accession No. P02746; UniProt Accession No. P02745; or UniProt Accession No. P02747), β2-glycoprotein I (β2GPI) and integrin αVβ3 / β5 (UniProt Accession No. P06756; UniProt Accession No. P05106; or UniProt Accession No. P18084). 70. A kit or composition according to any one of embodiments 57-58 and 61-66, wherein the pro-apoptotic signal binding agent comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of the following: phosphatidylserine, anti-annexin V antibody or its antigen binding fragment, annexin I (UniProt Accession No. P04083), calreticulin (UniProt Accession No. Q96L12), anti-CD36 antibody or its antigen binding fragment, thrombospondin-1 (TSP-1) (UniProt Accession No. P07996), anti-β2-glycoprotein I antibody or its antigen binding fragment, anti-milk fat globule-EGF-factor 8 (MFG-E8) or its antigen binding fragment, anti-phosphatidylserine receptor or its antigen binding fragment and anti-LDL receptor-related protein or its antigen binding fragment. 71. The kit or composition of any one of embodiments 57-70, comprising: c) Encapsulated nucleic acid. 72. The kit or composition of embodiment 71, wherein the nucleic acid is double-stranded DNA. 73. A kit or composition according to any one of embodiments 59-69 and 71-72, wherein the pro-apoptotic signal is annexin V. 74. A kit or composition according to any one of embodiments 57, 58, 61-66 and 70-72, wherein the pro-apoptotic signal binding agent is phosphatidylserine. 75. A kit or composition according to any one of embodiments 57, 58, 61-66 and 70-72, wherein the pro-apoptotic signal binding agent is an anti-Annexin V antibody or an antigen-binding fragment thereof. 76. A kit or composition according to any one of embodiments 59-69 and 71-72, wherein the pro-apoptotic signal is apo-15. 77. A kit or composition according to any of embodiments 57-76, wherein the hydrogel beads include artificial optical scattering properties that are substantially similar to corresponding optical scattering properties of target cells, and the artificial optical scattering properties are provided by: co-monomers, chemical side groups, encapsulated materials, colloidal silica, or the ratio of acrylamide to bisacrylamide. 78. A kit or composition according to any one of embodiments 57-77, wherein the hydrogel beads comprise a scattering modulating additive. 79. A kit or composition according to embodiment 77 or 78, wherein the optical scattering property that is substantially similar to the corresponding optical scattering property of the target cell is side scatter (SSC). 80. A kit or composition according to embodiment 77 or 78, wherein the optical scattering property that is substantially similar to the corresponding optical scattering property of the target cell is forward scatter (FSC). 81. A kit or composition according to any of embodiments 78-80, wherein the scattering modulation additive comprises a comonomer. 82. A kit or composition according to any of embodiments 78-80, wherein the scattering modulation additive comprises a suspension of nanoparticles. 83. The kit or composition of any one of embodiments 77-82, wherein the target cell is one of a lymphocyte, a monocyte, or a granulocyte. 84. The kit or composition of any one of embodiments 77-82, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell. 85. The kit or composition of any one of embodiments 77-82, wherein the target cell is a leukocyte. 86. The kit or composition of any one of embodiments 77-82, wherein the target cell is an immune cell. 87. The kit or composition of any one of embodiments 77-86, wherein the polymerized monomer is a biodegradable monomer. 88. The kit or composition of any one of embodiments 7-86, wherein the hydrogel beads are biodegradable. 89. The kit or composition of embodiment 87, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 90. The kit or composition of embodiment 88, wherein the hydrogel beads comprise a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 91. The kit or composition of embodiment 87, wherein the biodegradable monomer is a structured polysaccharide. 92. The kit or composition of embodiment 88, wherein the hydrogel beads comprise a structured polysaccharide. 93. A kit or composition according to embodiment 87, wherein the biodegradable monomer is selected from agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan polysaccharide, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp polysaccharide, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan , gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan and combinations thereof. 94. The kit or composition of embodiment 88, wherein the hydrogel beads comprise agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp laminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, Gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scissoran, sizoran, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof. 95. The kit or composition of any one of embodiments 73 and 77-94, wherein the annexin V is from a species selected from the group consisting of: human, rabbit, mouse, giant panda, Peruvian night monkey, minke whale, blue whale, Bosindicus x Bos taurus, zebu, yak, cattle, buffalo, common marmoset, Bactrian camel, Chinese native dog, goat, Philippine tarsier, American beaver, cattle, Panamanian capuchin, Canadian elk, red deer, Tarim Hara elk, beluga whale, Osso's kangaroo rat, desert flag-tailed kangaroo rat, Indian elephant, Przewalski's horse, gray whale, domestic cat, western gorilla, eastern gorilla, wolverine, gray seal, striped hyena, silvery gibbon, multi-striped ground squirrel, African jerboa, Pacific short-beaked dolphin, ring-tailed lemur, white-fin dolphin, African savanna elephant, long-tailed macaque, macaque , drill, yellow-bellied marmot, Alpine marmot, American marmot, forest musk deer, barking deer, pet mink, American ferret, Hawaiian monk seal, white-cheeked gibbon, raccoon dog, Pacific walrus, white-tailed deer Texas subspecies, killer whale, sheep, chimpanzee, East African baboon, Pacific pocket mouse, harbor seal, sperm whale, Ugandan red colobus monkey, Cluys's crested lemur, Svalbard reindeer, Yunnan golden monkey, Amazon squirrel monkey, East American squirrel, shrew, Eurasian wild boar, black leaf monkey, tree shrew, bottlenose dolphin, Arctic ground squirrel, polar bear, Arctic fox and California sea lion. 96. A kit or composition according to any one of embodiments 73 and 77-95, wherein the annexin V comprises a sequence that exhibits at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity to a sequence selected from SEQ ID NOs: 1-101. 97. The kit or composition of any one of embodiments 71-96, wherein the encapsulated nucleic acid is bound to a dye. 98. A kit or composition according to embodiment 97, wherein the dye bound to the encapsulated nucleic acid is selected from 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), DRAQ5 TM 、DRAQ7 TM 、CytoPhase TM Violet, Helix NP TM Blue, Helix NP TM Green, Helix NP TM NIR、YOYO TM -1.TOTO TM -1 iodide (Thermo Fisher Scientific), SYTOX TM Blue, ethidium bromide, SYBR TM Gold, SYBR TM Green, SYBR TM Safe, and crystal violet. 99. The kit or composition of embodiment 97, wherein the dye is 7AAD. 100. The kit or composition of embodiment 97, wherein the dye is propidium iodide. 101. The kit or composition of any one of embodiments 77-100, wherein the hydrogel beads have a refractive index greater than about 1.15. 102. The kit or composition of any one of embodiments 77-100, wherein the hydrogel beads have a refractive index greater than about 1.3. 103. The kit or composition of any one of embodiments 77-100, wherein the hydrogel beads have a refractive index greater than about 1.7. 104. The kit or composition of any one of embodiments 77-103, wherein the hydrogel beads have a diameter of less than about 100 μm. 105. The kit or composition of any one of embodiments 77-103, wherein the hydrogel beads have a diameter greater than about 10 μm. 106. The kit or composition of any one of embodiments 77-103, wherein the hydrogel beads have a diameter greater than about 1 μm. 107. A kit or composition according to any of embodiments 78-106, wherein the scattering modulation additive comprises polymer nanoparticles. 108. The kit or composition of embodiment 107, wherein the polymer nanoparticles comprise polystyrene. 109. The kit or composition of any one of embodiments 77-108, wherein the hydrogel beads are chemically functionalized hydrogel particles. 110. The kit or composition of any one of embodiments 77-109, wherein the hydrogel beads comprise free amine groups. 111. A kit or composition according to any one of embodiments 57, 58, 61-66 and 70-72, 74-75, 77-94 and 97-110, wherein the pro-apoptotic signal binding agent is attached to the free amine group. 112. The kit or composition of any one of embodiments 57-111, wherein the hydrogel beads comprise allylamine. 113. A kit or composition according to any one of embodiments 59-69, 71-73, 76-110 and 112, wherein when labeled with the pro-apoptotic signal, the hydrogel beads exhibit a mean fluorescence intensity (MFI) at least as high as the MFI of target cells labeled with the same pro-apoptotic signal. 114. A kit or composition according to any one of embodiments 59-69, 71-73, 76-110 and 112, wherein when labeled with the pro-apoptotic signal, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal. 115. A kit or composition according to any one of embodiments 57, 58, 61-66 and 70-72, 74-75, 77-94 and 97-112, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel beads exhibit a mean fluorescence intensity (MFI) that is at least as high as the MFI of target cells labeled with the same pro-apoptotic signal binding agent. 116. A kit or composition according to any one of embodiments 57, 58, 61-66 and 70-72, 74-75, 77-94 and 97-112, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as the MFI of target cells labeled with the same pro-apoptotic signal binding agent. 117. The kit or composition of embodiment 114 or 116, wherein the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%. 118. A kit or composition according to any one of embodiments 71-117, wherein when labeled with a DNA binding dye, the hydrogel beads exhibit a mean fluorescence intensity (MFI) that is at least as high as the MFI of target cells labeled with the same DNA binding dye. 119. A kit or composition according to any one of embodiments 71-117, wherein when labeled with a DNA binding dye, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same DNA binding dye. 120. The kit or composition of embodiment 119, wherein the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%. 121. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-56 or a population of hydrogel beads from a kit or composition according to any one of embodiments 57-120; b) contacting the hydrogel bead population with a pro-apoptotic signal and / or a DNA dye; c) measuring the concentration of pro-apoptotic signal and / or DNA dye in the hydrogel bead population; d) measuring the concentration of pro-apoptotic signal and / or DNA dye in the target cell sample; and e) comparing the concentration of pro-apoptotic signal and / or DNA dye measured in the hydrogel bead population with that in the target cell sample; Thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells. 122. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-56 or a population of hydrogel beads from a kit or composition according to any one of embodiments 57-120; b) contacting the hydrogel bead population with a pro-apoptotic signal and / or a DNA dye; c) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; d) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or DNA dye of the hydrogel beads; e) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pro-apoptotic cells. 123. A method for determining whether a target cell sample contains one or more dead cells or pro-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-56 or a population of hydrogel beads from a kit or composition according to any one of embodiments 57-120; wherein at least a population of hydrogel beads within the population of hydrogel beads comprises a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of pro-apoptotic signal and / or DNA dye in the hydrogel bead population; c) measuring the concentration of pro-apoptotic signal and / or DNA dye in the target cell sample; and d) comparing the concentration of pro-apoptotic signal and / or DNA dye measured in the hydrogel bead population with that in the target cell sample; Thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells. 124. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-56 or a population of hydrogel beads from a kit or composition according to any one of embodiments 57-120; wherein at least a population of hydrogel beads within the population of hydrogel beads comprises a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; c) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or DNA dye of the hydrogel beads; d) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pro-apoptotic cells. Additional embodiments

[0281] Notwithstanding the appended claims, the present disclosure sets forth the following additional numbered embodiments: 1. A hydrogel bead, comprising: a) polymerizable monomers and difunctional monomers; and b) Pro-apoptotic signal binders. 2. A hydrogel bead, comprising: a) polymerizable monomers and difunctional monomers; and b) Pro-apoptotic signals. 3. A hydrogel bead according to embodiment 1 or 2, wherein the pro-apoptotic signal comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to any one of the following: annexin V (UniProt Accession No. P08758), a polypeptide of SEQ ID NO: 103, an apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (UniProt Accession No. D9IWP9) (e.g., domain V), prothrombin or a fragment thereof (UniProt Accession No. P00734), milk fat globule-EGF-factor 8 (MFG-E8) (UniProt Accession No. Q08431), a phosphatidylserine receptor or a fragment thereof, SEQ ID NO: 102, CD36 (UniProt Accession No. P16671), LDL receptor-associated protein (UniProt Accession No. P01130), or an anti-calreticulin antibody or an antigen-binding fragment thereof. 3.1. The hydrogel bead according to Embodiment 3, wherein the pro-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof. 3.2. A hydrogel bead according to embodiment 4, wherein the phosphatidylserine receptor or fragment thereof comprises a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity with any one of the following: brain-specific angiogenesis inhibitory factor-1 (Bai1), Axl, Tyro3, ​​Mer, TIM-1 (also known as "KIM-1"), TIM-4, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), stabilizer-1, stabilizer-2, CD300a, CD300b, CD300f, receptor for advanced glycation end products (RAGE), complement component 1q (C1q), β2-glycoprotein I (β2GPI) and integrin αVβ3 / β5. 4. The hydrogel beads according to embodiment 1, wherein the pro-apoptotic signal binder is selected from phosphatidylserine, anti-annexin V antibody or its antigen binding fragment, annexin I, calreticulin, anti-CD36 antibody or its antigen binding fragment, thrombospondin-1 (TSP-1), anti-β2-glycoprotein I antibody or its antigen binding fragment, anti-milk fat globule-EGF-factor 8 (MFG-E8) or its antigen binding fragment, anti-phosphatidylserine receptor or its antigen binding fragment and anti-LDL receptor-associated protein or its antigen binding fragment. 5. The hydrogel bead according to any one of embodiments 1-4, comprising: c) Encapsulated nucleic acid. 6. The hydrogel bead of embodiment 5, wherein the nucleic acid is double-stranded DNA. 7. A hydrogel bead according to any one of embodiments 2-3 and 6, wherein the pro-apoptotic signal is annexin V. 8. A hydrogel bead according to any one of embodiments 2-3 and 6, wherein the pro-apoptotic signal binder is phosphatidylserine. 9. The hydrogel bead of any one of embodiments 1-3 and 6, wherein the pro-apoptotic signal binding agent is an anti-Annexin V antibody or an antigen-binding fragment thereof. 10. The hydrogel bead of any one of embodiments 1-3 and 6, wherein the pro-apoptotic signal is apo-15. 11. A hydrogel bead according to any one of embodiments 1-10, wherein the hydrogel bead comprises artificial optical scattering properties that are substantially similar to corresponding optical scattering properties of target cells, wherein the artificial optical scattering properties are provided by: comonomers, chemical side groups, encapsulated materials, colloidal silica, or a ratio of acrylamide to bisacrylamide. 12. The hydrogel bead of any one of embodiments 1-10, wherein the hydrogel bead comprises a scattering modulating additive. 13. The hydrogel bead of embodiment 11, wherein the optical scattering property substantially similar to the corresponding optical scattering property of the target cell is side scatter (SSC). 14. The hydrogel bead of embodiment 11, wherein the optical scattering property substantially similar to the corresponding optical scattering property of the target cell is forward scatter (FSC). 15. The hydrogel bead of any one of embodiments 11 or 12, wherein the scattering modulating additive comprises a comonomer. 16. The hydrogel bead of any one of embodiments 11 or 12, wherein the scattering modulating additive comprises a suspension of nanoparticles. 17. The hydrogel bead of any one of embodiments 11 and 13-16, wherein the target cell is one of a lymphocyte, a monocyte, or a granulocyte. 17.1 A hydrogel bead according to any one of embodiments 11 and 13-16, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell. 17.2 A hydrogel bead according to any one of embodiments 11 and 13-16, wherein the target cell is a leukocyte. 17.3 A hydrogel bead according to any one of embodiments 11 and 13-16, wherein the target cell is an immune cell. 18. The hydrogel bead of any one of embodiments 1-17, wherein the polymerized monomer is a biodegradable monomer. 18.1 The hydrogel bead of any one of embodiments 1-17, wherein the hydrogel is biodegradable. 19. The hydrogel bead of embodiment 18, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 19.1 A hydrogel bead according to embodiment 18, wherein the hydrogel comprises a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 20. The hydrogel bead of embodiment 18, wherein the biodegradable monomer is a structural polysaccharide. 20.1 The hydrogel bead of embodiment 18, wherein the hydrogel comprises a structural polysaccharide. 21. The hydrogel beads of embodiment 18, wherein the biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, Gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan and combinations thereof. 21.1 The hydrogel beads of embodiment 18, wherein the hydrogel comprises agar, agarose, alginic acid, polysaccharide acid, alpha glucan, amylopectin, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp laminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof. 22. A hydrogel bead according to any one of embodiments 7 and 11-22.1, wherein the annexin V is from a species selected from the group consisting of: human, rabbit, mouse, giant panda, Peruvian night monkey, minke whale, blue whale, zebu x Bos indicus x Bos taurus, zebu, yak, cattle, buffalo, common marmoset, Bactrian camel, Chinese native dog, goat, Philippine tarsier, American beaver, cattle, Panamanian capuchin, Canadian elk, red deer, Tarim Hara elk, beluga whale, Osso's kangaroo rat, desert flag-tailed kangaroo rat, Indian elephant, Przewalski's horse, gray whale, domestic cat, western gorilla, eastern gorilla, wolverine, gray seal, striped hyena, silvery gibbon, multi-striped ground squirrel, African jerboa, Pacific short-beaked dolphin, ring-tailed lemur, white-fin dolphin, African savanna elephant, long-tailed macaque, macaque , drill, yellow-bellied marmot, Alpine marmot, American marmot, forest musk deer, barking deer, pet mink, American ferret, Hawaiian monk seal, white-cheeked gibbon, raccoon dog, Pacific walrus, white-tailed deer Texas subspecies, killer whale, sheep, chimpanzee, East African baboon, Pacific pocket mouse, harbor seal, sperm whale, Ugandan red colobus monkey, Cluys's crested lemur, Svalbard reindeer, Yunnan golden monkey, Amazon squirrel monkey, East American squirrel, shrew, Eurasian wild boar, black leaf monkey, tree shrew, bottlenose dolphin, Arctic ground squirrel, polar bear, Arctic fox and California sea lion. 23. A hydrogel bead according to any one of embodiments 7 and 11-22, wherein the annexin V comprises a sequence that exhibits at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity to a sequence selected from SEQ ID NO: 101. 23.1. The hydrogel bead of any one of embodiments 7 and 11-22, wherein the annexin V comprises a sequence that exhibits at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity to the sequence of SEQ ID NO:1. 24. The hydrogel bead of any one of embodiments 5-23, wherein the encapsulated DNA is bound to a dye. 25. The hydrogel beads according to embodiment 24, wherein the dye bound to the encapsulated DNA is selected from 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), DRAQ5TM, DRAQ7TM, CytoPhaseTM Violet, Helix NPTM Blue, Helix NPTM Green, Helix NPTM NIR, YOYOTM-1, TOTOTM-1 iodide (Thermo Fisher Scientific), SYTOXTM Blue, ethidium bromide, SYBRTM Gold, SYBRTM Green, SYBRTM Safe, and crystal violet. 26. The hydrogel bead of embodiment 25, wherein the dye is 7AAD. 26.1 A hydrogel bead according to embodiment 25, wherein the dye is propidium iodide. 27. The hydrogel bead of any one of embodiments 1-26.1, wherein the hydrogel bead has a refractive index greater than about 1.15. 28. The hydrogel bead of any one of embodiments 1-26.1, wherein the hydrogel bead has a refractive index greater than about 1.3. 29. The hydrogel bead of any one of embodiments 1-26.1, wherein the hydrogel bead has a refractive index greater than about 1.7. 30. The hydrogel bead of any one of embodiments 1-29, wherein the hydrogel bead has a diameter of less than about 100 μm. 31. The hydrogel bead of any one of embodiments 1-29, wherein the hydrogel bead particles have a diameter greater than about 10 μm. 32. The hydrogel bead of any one of embodiments 1-29, wherein the hydrogel bead particles have a diameter greater than about 1 μm. 33. A hydrogel bead according to any one of embodiments 11-32, wherein the scattering modulation additive comprises polymer nanoparticles. 34. The hydrogel beads of embodiment 33, wherein the polymer nanoparticles comprise polystyrene. 35. The hydrogel bead of any one of embodiments 11-34, wherein the hydrogel bead is a chemically functionalized hydrogel particle. 36. The hydrogel bead of any one of embodiments 1-35, wherein the hydrogel bead comprises free amine groups. 36.1 The hydrogel bead of embodiment 36, wherein the apoptosis signal binding agent is attached to the free amine group. 37. The hydrogel bead of any one of embodiments 1-35, wherein the hydrogel bead comprises allylamine. 38. The hydrogel bead of any one of embodiments 1 and 3-37, wherein when labeled with the pro-apoptotic signal, the hydrogel exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal. 39. A hydrogel bead according to any one of embodiments 1 and 3-37, wherein when labeled with the pro-apoptotic signal, the hydrogel exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal. 40. The hydrogel bead of embodiment 39, wherein the MFI of the hydrogel and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%. 41. The hydrogel bead of any one of embodiments 5-40, wherein when labeled with the DNA binding dye, the hydrogel exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same DNA binding dye. 42. A hydrogel bead according to any one of embodiments 5-40, wherein when labeled with the pro-apoptotic signal, the hydrogel exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal. 43. A hydrogel bead according to embodiment 42, wherein the MFI of the hydrogel and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%. 44. A kit comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lacking a pro-apoptotic signal binding agent of said first hydrogel bead population; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 45. A composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lacking a pro-apoptotic signal binding agent of said first hydrogel bead population; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 46. ​​A composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) pro-apoptotic signals; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lack of pro-apoptotic signals of said first hydrogel bead population; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads. 47. The kit or composition of any one of embodiments 44-46, wherein the second hydrogel population and the third hydrogel population do not contain any nucleic acid. 48. The kit or composition of any one of embodiments 44-46, wherein the second hydrogel population and the third hydrogel population do not contain any double-stranded DNA. 49. The kit or composition of any one of embodiments 44-48, wherein the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a w / w ratio of about 1:1:1. 50. The kit or composition of any one of embodiments 44-48, wherein the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a ratio of about 1:1:1 by number of beads. 51. The kit or composition of any one of embodiments 44-48, wherein each of the first, second, and third hydrogel bead populations comprises approximately 10%-50% by weight of the total amount of hydrogel beads in the kit or composition. 52. The kit or composition of any one of embodiments 44-48, wherein each of the first, second, and third hydrogel bead populations comprises approximately 10%-50% of the total amount of hydrogel beads in the kit or composition, by number of beads. 53. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-43 or a population of hydrogel beads from a kit or composition according to any one of embodiments 44-52; b) contacting the hydrogel bead population with a pro-apoptotic signal and / or a DNA dye; c) measuring the concentration of pro-apoptotic signal and / or DNA dye in the hydrogel bead population; d) measuring the concentration of pro-apoptotic signal and / or DNA dye in the target cell sample; and e) comparing the concentration of pro-apoptotic signal and / or DNA dye measured in the hydrogel bead population with that in the target cell sample; Thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells. 54. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-43 or a population of hydrogel beads from a kit or composition according to any one of embodiments 44-52; b) contacting the hydrogel bead population with a pro-apoptotic signal and / or a DNA dye; c) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; d) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or DNA dye of the hydrogel beads; and e) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pro-apoptotic cells. 55. A method for determining whether a target cell sample contains one or more dead cells or pro-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-43 or a population of hydrogel beads from a kit or composition according to any one of embodiments 44-52; wherein at least a population of hydrogel beads within the hydrogel bead population comprises a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of pro-apoptotic signal and / or DNA dye in the hydrogel bead population; c) measuring the concentration of pro-apoptotic signal and / or DNA dye in the target cell sample; and d) comparing the concentration of pro-apoptotic signal and / or DNA dye measured in the hydrogel bead population with that in the target cell sample; Thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells. 56. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1-43 or a population of hydrogel beads from a kit or composition according to any one of embodiments 44-52; wherein at least a population of hydrogel beads within the hydrogel bead population comprises a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; c) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or DNA dye of the hydrogel beads; and d) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pro-apoptotic cells. Incorporated by Reference

[0282] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, any reference, article, publication, patent, patent publication and patent application cited herein is not and should not be regarded as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge of any country in the world. In addition, U.S. Patent No. 9,915,598, authorized on March 13, 2018 and entitled: Hydrogel Particles with Tunable Optical Properties, is hereby incorporated by reference for all purposes. In addition, U.S. Patent No. 9,714,897, authorized on July 25, 2017 and entitled: Hydrogel Particles with Tunable Optical Properties and Methods for Using the Same, is hereby incorporated by reference for all purposes. In addition, U.S. Patent No. 11,313,782, authorized on April 26, 2022 and entitled: Compositions and Methods for Cell-Like Calibration Particles, is hereby incorporated by reference for all purposes. In addition, U.S. Publication No. 2023 / 0067460, published on March 2, 2023 and entitled: Hydrogel Particles as Feeder Cells and Syntehtic Antigen Presenting Cells, is hereby incorporated by reference for all purposes. In addition, International Application No. PCT / US2023 / 066684, filed on May 5, 2023 and entitled: Engineered Particles as Red Blood Cell Mimics and Compositions Containing Same for Hematology, is hereby incorporated by reference for all purposes. In addition, International Publication No. WO 2021 / 226036, published on November 11, 2021 and entitled: Compositions and Methods for Passive Optical Barcoding for Multiplexed Assays, is hereby incorporated by reference for all purposes.

Claims

1. A hydrogel bead, comprising: a) polymerizable monomers and difunctional monomers; and b) Pro-apoptotic signal binders.

2. A hydrogel bead, comprising: a) polymerizable monomers and difunctional monomers; and b) Pro-apoptotic signals.

3. The hydrogel beads of claim 1 or 2, wherein the pro-apoptotic signal comprises annexin V, a polypeptide of SEQ ID NO: 103, an apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (e.g., domain V), prothrombin or a fragment thereof, milk fat globule-EGF-factor 8 (MFG-E8), a phosphatidylserine receptor or a fragment thereof, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 102, CD36, LDL receptor-associated protein, or an anti-calreticulin antibody or an antigen-binding fragment thereof. The hydrogel bead of claim 3 , wherein the pro-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof.

5. The hydrogel beads according to claim 4, wherein the phosphatidylserine receptor or fragment thereof is selected from brain-specific angiogenesis inhibitory factor-1 (Bai1), Axl, Tyro3, ​​Mer, TIM-1 (also known as "KIM-1"), TIM-4, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), stabilizer-1, stabilizer-2, CD300a, CD300b, CD300f, receptor for advanced glycation end products (RAGE), complement component 1q (C1q), β2-glycoprotein I (β2GPI) and integrin αVβ3 / β5.

6. The hydrogel beads according to claim 1, wherein the pro-apoptotic signal binder is selected from phosphatidylserine, anti-annexin V antibody or its antigen binding fragment, annexin I, calreticulin, anti-CD36 antibody or its antigen binding fragment, thrombospondin-1 (TSP-1), anti-β2-glycoprotein I antibody or its antigen binding fragment, anti-milk fat globule-EGF-factor 8 (MFG-E8) or its antigen binding fragment, anti-phosphatidylserine receptor or its antigen binding fragment and anti-LDL receptor-related protein or its antigen binding fragment.

7. The hydrogel bead according to any one of claims 1 to 6, comprising: c) encapsulated nucleic acid. The hydrogel bead of claim 7 , wherein the nucleic acid is double-stranded DNA.

9. The hydrogel bead according to any one of claims 2-3, 7 and 8, wherein the pro-apoptotic signal is Annexin V.

10. The hydrogel bead according to any one of claims 1, 3, 7 and 8, wherein the pro-apoptotic signal binding agent is phosphatidylserine.

11. The hydrogel bead according to any one of claims 1, 3, 7 and 8, wherein the pro-apoptotic signal binding agent is an anti-Annexin V antibody or an antigen-binding fragment thereof.

12. The hydrogel bead according to any one of claims 2-3, 7 and 8, wherein the pro-apoptotic signal is apo-15.

13. The hydrogel bead of any one of claims 1-12, wherein the hydrogel bead comprises artificial optical scattering properties substantially similar to corresponding optical scattering properties of target cells, the artificial optical scattering properties being provided by: comonomers, chemical side groups, encapsulated materials, colloidal silica, or a ratio of acrylamide to bisacrylamide.

14. The hydrogel bead of any one of claims 1-13, wherein the hydrogel bead comprises a scattering modulating additive.

15. The hydrogel bead of claim 13, wherein the optical scattering property that is substantially similar to a corresponding optical scattering property of the target cell is side scatter (SSC).

16. The hydrogel bead of claim 13, wherein the optical scattering property that is substantially similar to a corresponding optical scattering property of the target cell is forward scatter (FSC).

17. The hydrogel bead of claim 14, wherein the scattering modulating additive comprises a comonomer.

18. The hydrogel bead of claim 14, wherein the scattering modulating additive comprises a suspension of nanoparticles.

19. The hydrogel bead according to any one of claims 13 and 15-18, wherein the target cell is one of a lymphocyte, a monocyte or a granulocyte.

20. The hydrogel bead according to any one of claims 13 and 15-18, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell.

21. The hydrogel bead of any one of claims 13 and 15-18, wherein the target cell is a leukocyte.

22. The hydrogel bead of any one of claims 13 and 15-18, wherein the target cell is an immune cell.

23. The hydrogel bead of any one of claims 1-22, wherein the polymerized monomer is a biodegradable monomer.

24. The hydrogel bead of any one of claims 1-22, wherein the hydrogel bead is biodegradable.

25. The hydrogel bead of claim 23, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain.

26. The hydrogel bead of claim 23, wherein the hydrogel bead comprises a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain.

27. The hydrogel bead of claim 23, wherein the biodegradable monomer is a structural polysaccharide.

28. The hydrogel bead of claim 23, wherein the hydrogel bead comprises a structural polysaccharide.

29. The hydrogel beads of claim 23, wherein the biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, Gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan and combinations thereof.

30. The hydrogel beads of claim 23, wherein the hydrogel beads comprise agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp laminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof.

31. The hydrogel bead of any one of claims 9 and 13-30, wherein the annexin V is from a species selected from the group consisting of: human, rabbit, mouse, giant panda (Ailuropoda melanoleuca), Peruvian night monkey (Aotus nancymaae), minke whale (Balaenoptera acutorostrata scammoni), blue whale (Balaenoptera musculus), zebu x cattle (Bos indicus x Bos taurus), zebu (Bos indicus), yak (Bos mutus), cattle (Bostaurus), buffalo (Bubalus bubalis), common marmoset (Callithrix jacchus), Bactrian camel (Camelus bactrianus), Chinese pastoral dog (Canis lupus familiaris), goat (Capra hircus), Philippine tarsier (Carlito syrichta), American beaver (Castor canadensis), cattle, Panamanian capuchin monkey (Cebusimitator), Canadian elk (Cervus canadensis), red deer (Cervus elaphus), Tarim Hara red deer (Cervus hanglu yarkandensis), beluga whale (Delphinapterus leucas), Austrian kangaroo rat (Dipodomys ordii), desert flag-tailed kangaroo rat (Dipodomys spectabilis), Indian elephant (Elephasmaximus indicus), Przewalski's horse (Equus przewalskii), gray whale (Eschrichtius robustus), domestic cat (Felis catus), western gorilla (Gorilla gorilla), eastern gorilla (Gorilla beringer), mink (Gulo gulo luscus), gray seal (Halichoerus grypus), striped hyena (Hyaena hyaena), silvery gibbon (Hylobates moloch), multi-striped ground squirrel (Ictidomys tridecemlineatus), African jerboa (Jaculus jaculus), Pacific white-necked dolphin (Lagenorhynchus obliquidens), ring-tailed lemur (Lemur catta), white-fin dolphin (Lipotes vexillifer), African savanna elephant (Loxodontaafricana), long-tailed macaque (Macacafascicularis), macaque (Macaca mulatta), drill (Mandrillus leucophaeus), yellow-bellied marmot (Marmota flaviventris), Alpine marmot (Marmota marmota marmota), American marmot (Marmotamonax), forest musk deer (Moschus berezovskii), barking deer (Muntiacus muntjak), pet mink (Mustelaputorius furo), American water mink (Neogale vison), Hawaiian monk seal (Neomonachusschauinslandi), white-cheeked gibbon (Nomascus leucogenys), raccoon dog (Nyctereutes procyonoides), Pacific walrus (Odobenus rosmarus divergens), Texas subspecies of white-tailed deer (Odocoileusvirginianus texanus), killer whale (Orcinus orca), sheep (Ovis aries), chimpanzees (Pantroglodytes), East African baboons (Papio anubis), Pacific pocket mice (Perognathus longimembrispacificus), harbor seals (Phoca vitulina), sperm whales (Physeter catodon), Ugandan red colobus monkeys (Piliocolobus tephrosceles), Crow's lemurs (Propithecus coquereli), Svalbard reindeer (Rangifer tarandus platyrhyncus), Yunnan snub-nosed monkeys (Rhinopithecus bieti), Amazon squirrel monkeys (Saimiri boliviensis boliviensis), Eastern American squirrels (Sciurus carolinensis), shrews (Sorexaraneus), Eurasian wild boars (Sus scrofa), black leaf monkeys (Trachypithecus francoisi), tree shrews (Tupaiachinensis), bottlenose dolphins (Tursiops truncatus), Arctic ground squirrel (Urocitellus parryii), polar bear (Ursus maritimus), Arctic fox (Vulpes lagopus), and California sea lion (Zalophus californianus).

32. The hydrogel bead of any one of claims 13 and 13-31, wherein the Annexin V comprises a sequence that exhibits at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity to a sequence selected from SEQ ID NOs: 1-101.

33. The hydrogel bead of any one of claims 7-32, wherein the encapsulated nucleic acid is bound to a dye.

34. The hydrogel bead of claim 33, wherein the dye bound to the encapsulated nucleic acid is selected from the group consisting of 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), DRAQ5 TM 、DRAQ7 TM 、CytoPhase TM Violet, Helix NP TM Blue、HelixNP TM Green, Helix NP TM NIR、YOYO TM -1.TOTO TM -1 Iodide (Thermo Fisher Scientific), TO-PRO- SYTOX TM Blue, ethidium bromide, SYBR TM Gold, SYBR TM Green, SYBR TM Safe, and crystal violet.

35. The hydrogel bead of claim 34, wherein the dye is 7AAD.

36. The hydrogel bead of claim 34, wherein the dye is propidium iodide.

37. The hydrogel beads of any one of claims 1-36, wherein the hydrogel beads have a refractive index greater than about 1.

15.

38. The hydrogel beads of any one of claims 1-36, wherein the hydrogel beads have a refractive index greater than about 1.

3.

39. The hydrogel beads of any one of claims 1-36, wherein the hydrogel beads have a refractive index greater than about 1.

7.

40. The hydrogel bead of any one of claims 1-39, wherein the hydrogel bead has a diameter of less than about 100 μm.

41. The hydrogel bead of any one of claims 1-39, wherein the hydrogel bead has a diameter greater than about 10 μm.

42. The hydrogel bead of any one of claims 1-39, wherein the hydrogel bead has a diameter greater than about 1 μm.

43. The hydrogel bead of any one of claims 14-42, wherein the scattering modulating additive comprises polymer nanoparticles.

44. The hydrogel beads of claim 43, wherein the polymer nanoparticles comprise polystyrene.

45. The hydrogel beads of any one of claims 1-44, wherein the hydrogel beads are chemically functionalized hydrogel particles.

46. ​​The hydrogel bead of any one of claims 1-45, wherein the hydrogel bead comprises free amine groups.

47. The hydrogel bead of claim 46, wherein the pro-apoptotic signal binding agent is attached to the free amine group.

48. The hydrogel beads of any one of claims 1-47, wherein the hydrogel beads comprise allylamine.

49. The hydrogel bead of any one of claims 2-5, 7-9, and 12-48, wherein when labeled with the pro-apoptotic signal, the hydrogel bead exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal.

50. The hydrogel bead of any one of claims 2-5, 7-9, and 12-48, wherein when labeled with the pro-apoptotic signal, the hydrogel bead exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal.

51. The hydrogel bead of any one of claims 1, 6-8, 10-11, and 13-48, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel bead exhibits a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal binding agent.

52. The hydrogel bead of any one of claims 1, 6-8, 10-11, and 13-48, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel bead exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal binding agent.

53. The hydrogel bead of claim 50 or 52, wherein the MFI of the hydrogel bead and the MFI of the target cell are within 50%, 40%, 30%, 20% or 10%.

54. The hydrogel bead of any one of claims 7-53, wherein when labeled with a DNA binding dye, the hydrogel bead exhibits a mean fluorescence intensity (MFI) at least as high as that of a target cell labeled with the same DNA binding dye.

55. The hydrogel bead of any one of claims 7-53, wherein when labeled with a DNA binding dye, the hydrogel bead exhibits a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same DNA binding dye.

56. The hydrogel bead of claim 55, wherein the MFI of the hydrogel bead and the MFI of the target cell are within 50%, 40%, 30%, 20% or 10%.

57. A kit, comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) a pro-apoptotic signal binder; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) a pro-apoptotic signal binder; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lacking a pro-apoptotic signal binding agent in said first population of hydrogel beads; and iii) lacking encapsulated nucleic acids of said first population of hydrogel beads.

58. A composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) a pro-apoptotic signal binder; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) a pro-apoptotic signal binder; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lacking a pro-apoptotic signal binding agent in said first population of hydrogel beads; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads.

59. A kit comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerized monomers; ii) pro-apoptotic signals; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lack of pro-apoptotic signals of said first population of hydrogel beads; and iii) lacking encapsulated nucleic acids of said first population of hydrogel beads.

60. A composition comprising: a) a first population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) pro-apoptotic signals; and iii) encapsulated nucleic acid; b) a second population of hydrogel beads, each bead comprising: i) polymerizable monomers; ii) pro-apoptotic signals; but iii) lacking encapsulated nucleic acids from said first population of hydrogel beads; and c) a third hydrogel bead population, the third hydrogel bead population comprising: i) polymerized monomers; but ii) lack of pro-apoptotic signals of said first hydrogel bead population; but iii) lacking encapsulated nucleic acids of said first population of hydrogel beads.

61. The kit or composition of any one of claims 57-60, wherein the second hydrogel population and the third hydrogel population are free of any nucleic acid.

62. The kit or composition of any one of claims 57-60, wherein the second hydrogel population and the third hydrogel population do not contain any double-stranded DNA.

63. The kit or composition of any one of claims 57-62, wherein the first hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population are in a w / w ratio of about 1:1:

1.

64. The kit or composition of any one of claims 57-62, wherein the first, second, and third hydrogel bead populations are in a ratio of about 1:1:1 by number of beads.

65. The kit or composition of any one of claims 57-62, wherein each of the first, second, and third hydrogel bead populations comprises about 10%-50% by weight of the total amount of hydrogel beads in the kit or composition.

66. The kit or composition of any one of claims 57-62, wherein each of the first, second, and third hydrogel bead populations comprises about 10%-50% of the total amount of hydrogel beads in the kit or composition, by number of beads.

67. The kit or composition of any one of claims 59-66, wherein the pro-apoptotic signal comprises annexin V, a polypeptide of SEQ ID NO: 103, an apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (e.g., domain V), prothrombin or a fragment thereof, milk fat globule-EGF-factor 8 (MFG-E8), a phosphatidylserine receptor or a fragment thereof, a polypeptide having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity to SEQ ID NO: 102, CD36, LDL receptor-related protein, or an anti-calreticulin antibody or an antigen-binding fragment thereof.

68. The kit or composition of any one of claims 59-66, wherein the pro-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof.

69. The kit or composition of claim 68, wherein the phosphatidylserine receptor or fragment thereof is selected from the group consisting of brain-specific angiogenesis inhibitory factor-1 (Bai1), Axl, Tyro3, ​​Mer, TIM-1 (also known as "KIM-1"), TIM-4, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), stabilizer-1, stabilizer-2, CD300a, CD300b, CD300f, receptor for advanced glycation end products (RAGE), complement component 1q (C1q), β2-glycoprotein I (β2GPI) and integrin αVβ3 / β5.

70. The kit or composition of any one of claims 57-58 and 61-66, wherein the pro-apoptotic signal binding agent is selected from phosphatidylserine, anti-annexin V antibody or antigen binding fragment thereof, annexin I, calreticulin, anti-CD36 antibody or antigen binding fragment thereof, thrombospondin-1 (TSP-1), anti-β2-glycoprotein I antibody or antigen binding fragment thereof, anti-milk fat globule-EGF-factor 8 (MFG-E8) or antigen binding fragment thereof, anti-phosphatidylserine receptor or antigen binding fragment thereof and anti-LDL receptor-related protein or antigen binding fragment thereof.

71. The kit or composition according to any one of claims 57-70, comprising: c) Encapsulated nucleic acid.

72. The kit or composition of claim 71, wherein the nucleic acid is double-stranded DNA.

73. The kit or composition of any one of claims 59-69 and 71-72, wherein the pro-apoptotic signal is Annexin V.

74. The kit or composition of any one of claims 57, 58, 61-66, and 70-72, wherein the pro-apoptotic signal binding agent is phosphatidylserine.

75. The kit or composition of any one of claims 57, 58, 61-66, and 70-72, wherein the pro-apoptotic signal binding agent is an anti-Annexin V antibody or an antigen-binding fragment thereof.

76. The kit or composition of any one of claims 59-69 and 71-72, wherein the pro-apoptotic signal is apo-15.

77. The kit or composition of any one of claims 57-76, wherein the hydrogel beads comprise artificial optical scattering properties substantially similar to corresponding optical scattering properties of target cells, the artificial optical scattering properties being provided by: comonomers, chemical side groups, encapsulated materials, colloidal silica, or a ratio of acrylamide to bisacrylamide.

78. The kit or composition of any one of claims 57-77, wherein the hydrogel beads comprise a scattering modulating additive.

79. The kit or composition of claim 77 or 78, wherein the optical scattering property that is substantially similar to the corresponding optical scattering property of the target cell is side scatter (SSC).

80. The kit or composition of claim 77 or 78, wherein the optical scattering property that is substantially similar to the corresponding optical scattering property of the target cell is forward scatter (FSC).

81. The kit or composition of any one of claims 78-80, wherein the scattering modulation additive comprises a comonomer.

82. The kit or composition of any one of claims 78-80, wherein the scattering modulating additive comprises a suspension of nanoparticles.

83. The kit or composition of any one of claims 77-82, wherein the target cell is one of a lymphocyte, a monocyte, or a granulocyte.

84. The kit or composition of any one of claims 77-82, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell.

85. The kit or composition of any one of claims 77-82, wherein the target cell is a leukocyte.

86. The kit or composition of any one of claims 77-82, wherein the target cell is an immune cell.

87. The kit or composition of any one of claims 77-86, wherein the polymerized monomer is a biodegradable monomer.

88. The kit or composition of any one of claims 7-86, wherein the hydrogel beads are biodegradable.

89. The kit or composition of claim 87, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain.

90. The kit or composition of claim 88, wherein the hydrogel beads comprise a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain.

91. The kit or composition of claim 87, wherein the biodegradable monomer is a structural polysaccharide.

92. The kit or composition of claim 88, wherein the hydrogel beads comprise a structured polysaccharide.

93. The kit or composition of claim 87, wherein the biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan , gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scizolan, sizolan, welan gum, xanthan gum, xylan, xyloglucan, zymosan and combinations thereof.

94. The kit or composition of claim 88, wherein the hydrogel beads comprise agar, agarose, alginic acid, polysaccharide acid, alpha glucan, pullulan, amylose, arabinoxylan, beta glucan, callose, capsullan, carrageenan, cellodextrin, animal cellulose, cellulose, chitin, chitosan, kelp, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, polysucrose, levan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, Gellan gum, dextran, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hydroxypropyl methylcellulose, icodextrin, inulin, kefir polysaccharide, laminarin, lentinan, levan polysaccharide, lichen polysaccharide, mannan, mixed-linked glucan, paramylon, pectic acid, pectin, pentameric starch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, scissoran, sizoran, welan gum, xanthan gum, xylan, xyloglucan, zymosan or a combination thereof.

95. The kit or composition of any one of claims 73 and 77-94, wherein the annexin V is from a species selected from the group consisting of humans, rabbits, mice, giant pandas, Peruvian night monkeys, minke whales, blue whales, zebu x cattle, zebu, yak, cattle, buffalo, common marmosets, Bactrian camels, Chinese pastoral dogs, goats, Philippine tarsiers, American beavers, cattle, Panamanian capuchins, Canadian elk, red deer, Tarim Hara elk, beluga whales, Osso's kangaroo rats, desert flag-tailed kangaroo rats, Indian elephants, Przewalski's horses, gray whales, domestic cats, western gorillas, eastern gorillas, martens, gray seals, striped hyenas, silvery gibbons, and ground squirrels. , African jerboa, Pacific short-necked dolphin, ring-tailed lemur, white-fin dolphin, African savanna elephant, long-tailed macaque, macaque, drill, yellow-bellied marmot, Alpine marmot, American marmot, forest musk deer, barking deer, pet mink, American water ferret, Hawaiian monk seal, white-cheeked gibbon, raccoon dog, Pacific walrus, white-tailed deer Texas subspecies, killer whale, sheep, chimpanzee, East African baboon, Pacific pocket mouse, harbor seal, sperm whale, Ugandan red colobus monkey, Cluvist's crested lemur, Svalbard reindeer, Yunnan snub-nosed monkey, Amazon squirrel monkey, Eastern American squirrel, shrew, Eurasian wild boar, dark leaf monkey, tree shrew, bottlenose dolphin, Arctic ground squirrel, polar bear, Arctic fox and California sea lion.

96. The kit or composition of any one of claims 73 and 77-95, wherein the Annexin V comprises a sequence that exhibits at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity to a sequence selected from SEQ ID NOs: 1-101.

97. The kit or composition of any one of claims 71-96, wherein the encapsulated nucleic acid is bound to a dye.

98. The kit or composition of claim 97, wherein the dye bound to the encapsulated nucleic acid is selected from 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), DRAQ5 TM 、DRAQ7 TM 、CytoPhase TM Violet, Helix NP TM Blue, Helix NP TM Green, Helix NP TM NIR、YOYO TM -1.TOTO TM -1 Iodide (Thermo FisherScientific), TO-PRO- SYTOX TM Blue, ethidium bromide, SYBR TM Gold, SYBR TM Green, SYBR TM Safe, and crystal violet.

99. The kit or composition of claim 97, wherein the dye is 7AAD.

100. The kit or composition of claim 97, wherein the dye is propidium iodide.

101. The kit or composition of any one of claims 77-100, wherein the hydrogel beads have a refractive index greater than about 1.

15.

102. The kit or composition of any one of claims 77-100, wherein the hydrogel beads have a refractive index greater than about 1.

3.

103. The kit or composition of any one of claims 77-100, wherein the hydrogel beads have a refractive index greater than about 1.

7.

104. The kit or composition of any one of claims 77-103, wherein the hydrogel beads have a diameter of less than about 100 μm.

105. The kit or composition of any one of claims 77-103, wherein the hydrogel beads have a diameter greater than about 10 μm.

106. The kit or composition of any one of claims 77-103, wherein the hydrogel beads have a diameter greater than about 1 μm.

107. The kit or composition of any one of claims 78-106, wherein the scattering modulating additive comprises polymer nanoparticles.

108. The kit or composition of claim 107, wherein the polymer nanoparticles comprise polystyrene.

109. The kit or composition of any one of claims 77-108, wherein the hydrogel beads are chemically functionalized hydrogel particles.

110. The kit or composition of any one of claims 77-109, wherein the hydrogel beads comprise free amine groups.

111. The kit or composition of any one of claims 57, 58, 61-66 and 70-72, 74-75, 77-94 and 97-110, wherein the pro-apoptotic signal binding agent is attached to the free amine group.

112. The kit or composition of any one of claims 57-111, wherein the hydrogel beads comprise allylamine.

113. The kit or composition of any one of claims 59-69, 71-73, 76-110 and 112, wherein when labeled with the pro-apoptotic signal, the hydrogel beads exhibit a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal.

114. The kit or composition of any one of claims 59-69, 71-73, 76-110 and 112, wherein when labeled with the pro-apoptotic signal, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal.

115. The kit or composition of any one of claims 57, 58, 61-66 and 70-72, 74-75, 77-94 and 97-112, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel beads exhibit a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same pro-apoptotic signal binding agent.

116. The kit or composition of any one of claims 57, 58, 61-66 and 70-72, 74-75, 77-94 and 97-112, wherein when labeled with the pro-apoptotic signal binding agent, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same pro-apoptotic signal binding agent.

117. The kit or composition of claim 114 or 116, wherein the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%.

118. The kit or composition of any one of claims 71-117, wherein when labeled with a DNA binding dye, the hydrogel beads exhibit a mean fluorescence intensity (MFI) at least as high as that of target cells labeled with the same DNA binding dye.

119. The kit or composition of any one of claims 71-117, wherein when labeled with a DNA binding dye, the hydrogel beads exhibit a mean fluorescence intensity (MFI) substantially the same as that of target cells labeled with the same DNA binding dye.

120. The kit or composition of claim 119, wherein the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%.

121. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of claims 1-56 or a population of hydrogel beads from a kit or composition according to any one of claims 57-120; b) contacting the hydrogel bead population with a pro-apoptotic signal and / or a DNA dye; c) measuring the concentration of pro-apoptotic signal and / or DNA dye in the hydrogel bead population; d) measuring the concentration of pro-apoptotic signal and / or DNA dye in the target cell sample; and e) comparing the concentration of pro-apoptotic signal and / or DNA dye measured in the hydrogel bead population with that in the target cell sample; Thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

122. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of claims 1-56 or a population of hydrogel beads from a kit or composition according to any one of claims 57-120; b) contacting the hydrogel bead population with a pro-apoptotic signal and / or a DNA dye; c) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; d) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or DNA dye of the hydrogel beads; e) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pro-apoptotic cells.

123. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of claims 1-56 or a population of hydrogel beads from a kit or composition according to any one of claims 57-120; wherein at least the hydrogel bead population within the hydrogel bead population comprises a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of pro-apoptotic signal and / or DNA dye in the hydrogel bead population; c) measuring the concentration of pro-apoptotic signal and / or DNA dye in the target cell sample; and d) comparing the concentration of pro-apoptotic signal and / or DNA dye measured in the hydrogel bead population with that in the target cell sample; Thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

124. A method for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of claims 1-56 or a population of hydrogel beads from a kit or composition according to any one of claims 57-120; wherein at least the hydrogel bead population within the hydrogel bead population comprises a pro-apoptotic signal and / or a DNA dye; b) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the hydrogel bead population in a cell counting device; c) calibrating the cell counting device based on the measured concentration of the pro-apoptotic signal and / or DNA dye of the hydrogel beads; d) measuring the concentration of the pro-apoptotic signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead cells or pro-apoptotic cells.

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