Apoptosis cell mimetic

Abstract

Hydrogel beads that mimic live cells, dead cells, and apoptotic cells are provided herein. The disclosure also provides kits and compositions of the hydrogel beads. The 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. The invention provides, for example, hydrogel beads comprising a) a polymerizing monomer and a bifunctional monomer, and b) a pre-apoptosis signal binder.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 348,414, filed on June 2, 2022. The foregoing application is hereby incorporated by reference in its entirety for all purposes.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (SLIN_012_01WO_SeqList_ST26.xml; size: 124,536 bytes; created on June 2, 2023) are hereby incorporated by reference in their entirety.

[0003] Field The present disclosure generally relates to hydrogel beads that mimic 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] Background Flow cytometry is used to analyze and detect the chemical and physical properties of cells. Data from this technique enables physicians to diagnose and stage multiple diseases, including cancer. Removing dead and dying cells from flow cytometry data is important to ensure the accuracy of the analysis. Dead cells are autofluorescent and difficult to exclude from the analysis based on forward and side scatter alone. In flow cytometry viability assays, cells are stained with a viability dye 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 cell type of interest. Obtaining these purified cells using heating or chemical methods is an uneconomical, time-consuming process that is not well standardized and prone to batch-to-batch variation. Additionally, the 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, live, and dying cells. Summary of the Invention Means for Solving the Problems

[0005] Summary of the Invention Hydrogel beads are provided herein that comprise a) a polymerizable monomer and a bifunctional monomer, and b) a pre-apoptotic signal binder.

[0006] Hydrogel beads are provided herein that comprise a) a polymerizable monomer and a bifunctional monomer, and b) a pre-apoptotic signal.

[0007] Hydrogel beads are provided herein that comprise a) a polymerizable monomer and a bifunctional monomer, b) a pre-apoptotic signal binder, and c) an encapsulated nucleic acid.

[0008] Hydrogel beads are provided herein that comprise: a) a polymerizable monomer and a bifunctional monomer; b) a pre-apoptosis signal; and c) an encapsulated nucleic acid.

[0009] A kit is provided herein that comprises: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer; ii) a pre-apoptosis signal binder; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer; and ii) a pre-apoptosis signal binder, but lacks the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer, but lacks the pre-apoptosis signal binder of the first population of hydrogel beads and lacks the encapsulated nucleic acid of the first population of hydrogel beads.

[0010] A composition is provided herein that comprises: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer; ii) a pre-apoptosis signal binder; and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer; and ii) a pre-apoptosis signal binder, but lacks the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer, but lacks the pre-apoptosis signal binder of the first population of hydrogel beads and lacks the encapsulated nucleic acid of the first population of hydrogel beads.

[0011] A kit comprising: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer, ii) a pre-apoptosis signal, and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer and ii) a pre-apoptosis signal, but lacks the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, which comprises: i) a polymerizable monomer, but lacks the pre-apoptosis signal of the first population of hydrogel beads and ii) the encapsulated nucleic acid of the first population of hydrogel beads, is provided herein.

[0012] A composition comprising: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer, ii) a pre-apoptosis signal, and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer and ii) a pre-apoptosis signal, but lacks the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, which comprises: i) a polymerizable monomer, but lacks the pre-apoptosis signal of the first population of hydrogel beads and ii) the encapsulated nucleic acid of the first population of hydrogel beads, is provided herein.

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

[0014] 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 as disclosed herein, or a population of hydrogel beads from a kit or composition as disclosed herein; b) contacting the population of hydrogel beads 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 population of hydrogel beads with a cytometry device; d) calibrating the cytometry 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. A method is provided herein.

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

[0016] 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 as disclosed herein, or a population of hydrogel beads from a kit or composition as disclosed herein, wherein at least a sub-population of the hydrogel beads within the population of hydrogel beads 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 population of hydrogel beads with a cytometry device; c) calibrating the cytometry device based on the measured concentration of the pre-apoptotic signal and / or the DNA dye of the hydrogel beads; and d) 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. A method is provided herein. Brief Description of the Drawings

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[0057] Detailed Description of the Invention The present disclosure provides hydrogel beads (Section IV), as well as kits (Section VI) and compositions (Section VII) that include them. The present disclosure also provides methods of using hydrogel beads (Section VIII), as well as kits and compositions that include them.

[0058] The hydrogel beads provided herein mimic live cells, dead cells, or apoptotic cells. These hydrogel beads have light scattering properties (e.g., forward scatter and / or side scatter) that can be adjusted to match those of a target cell population. The properties of these beads are further described in Section V.

[0059] Advantageously, these hydrogel beads, as well as compositions and kits containing the same, can be used to determine whether a target cell population contains live cells, dead cells, and / or cells undergoing apoptosis that are not yet dead. Furthermore, hydrogel beads, as well as compositions and kits containing the same, can be used to quantify the number of live cells, dead cells, and / or apoptotic cells in a target cell population. Compositions containing hydrogel beads are superior to cell-containing compositions for several reasons. First, the number of hydrogel beads serving as live cell controls, dead cell controls, and apoptotic cell controls can be adjusted. In contrast, the amounts of dead cells, live cells, and apoptotic cells in a cell population serving as a control for apoptosis cannot be precisely controlled. Thus, a hydrogel bead composition can be generated that has 33% beads each serving as a dead cell mimic, a live cell mimic, and an apoptotic cell mimic. Second, compositions containing hydrogel beads stained with pre-apoptotic signals and viability dyes show distinct positive and negative bead populations. In contrast, the separation between positive and negative cell populations is not as distinct. Third, compositions containing hydrogel beads show less variability than cells. Different lots of cells can exhibit different properties depending on the age of the cells, whereas hydrogel bead compositions are stable for at least 37 days. Fourth, the use of hydrogel bead compositions is less time-consuming than using a cell population as a control for apoptosis because the hydrogel bead composition requires neither cell culture nor induction of apoptosis. In contrast, when using a cell population as a control for apoptosis, apoptosis must be induced in the control cells using a heating method or a chemical method. This process is time-consuming, uneconomical, and not well standardized.

[0060] I. Definitions 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.

[0061] The terms "at least one" and "one or more" are used interchangeably to mean that an article can include one or more of the recited elements.

[0062] As used herein, the term "about" refers to ±10% of the recited number, unless otherwise specified or apparent from the context, and except where such a range would exceed 100% of the possible value or be less than 0% of the possible value, e.g., a content less than 0% of a component or greater than 100% of the total content of a composition. For example, a reference to about 10% monomer by weight of a hydrogel means that the monomer can be present in any amount in the range of 9% to 11% by weight of the hydrogel. It is to be understood that all numbers expressing amounts, ratios, and numerical properties of components, reaction conditions, etc., used herein and in the claims are intended to be modified in all instances by the term "about" unless otherwise indicated.

[0063] The term "scattering modulating additive" refers to any element that can modulate the side scattering of hydrogel beads. Non-limiting examples of scattering modulating additives include nanoparticles made of polymethylmethacrylate (PMMA), polystyrene (PS), or silica, and / or high refractive index molecules added to the hydrogel such as alkyl acrylates, alkyl methacrylates, vinyls, enes (such as styrene and methylstyrene), etc., optionally substituted on the aromatic ring with an alkyl group (such as methyl, ethyl, or tert-butyl) or a halogen (such as chlorostyrene).

[0064] The term "hydrogel beads" refers to particles made from a hydrogel material and optionally including one or more additional elements for use in the recited cytometry assay or Coulter assay. In some embodiments, the hydrogels of the present disclosure are substantially spherical in shape and can resemble one or more target cells.

[0065] The term "light scattering characteristics" refers to the forward scatter (FSC) and side scatter (SSC) characteristics of cells or hydrogels.

[0066] The term "dead cell" refers to non-viable cells. In some embodiments, dead cells have a permeable, ruptured, or non-existent membrane such that the cytoplasm and nucleus are accessible by one or more viability dyes / markers.

[0067] The term "pre-apoptotic cell" refers to a cell in which apoptosis has been induced but which is not yet dead.

[0068] The term "hydrogel" refers to a material comprising a polymeric three-dimensional network that swells in the presence of water (i.e., "wet state") and shrinks in the absence of water (or by reduction in the amount of water) (i.e., "dehydrated state") but does not dissolve in water. As used herein, the term "hydrogel" refers to the material in its wet or dehydrated state. Swelling or water absorption is the result of the presence of hydrophilic functional groups attached to or dispersed within the polymeric network. Crosslinking between adjacent polymers results in the water-insolubility of these hydrogels. Crosslinking can result from chemical (i.e., covalent) or physical (i.e., van der Waals forces, hydrogen bonding, ionic force, etc.) bonds. These chemical crosslinks can also be hydrolyzed under certain conditions to reverse the insolubility of the hydrogel. Multiple 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.

[0069] The term "bifunctional monomer" refers to a monomer containing a first functional group and a second functional group, where the first functional group polymerizes 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.

[0070] The term "forward scatter" refers to the light scattering properties of a material measured in the parallel direction of the propagation of light. Forward scatter is a general measure of the size of particles and can also be affected by the refractive index of the hydrogels of the present disclosure.

[0071] The term "side scatter" refers to the light scattering properties of a material when measured in the perpendicular direction of the propagation of light. Side scatter is a general measure of the complexity of particles and can also be affected by the refractive index of the hydrogels of the present disclosure.

[0072] The term "substantially similar" refers to 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.

[0073] The term "cytometry device" refers to a device used to measure the number and characteristics of cells. Variables that can be measured by cytometry methods include cell size, cell number, cell morphology (shape and structure), cell cycle phase, DNA content, and the presence or absence of specific proteins on the cell surface or in the cytoplasm. A general cytometry 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.

[0074] The term "antigen-binding fragment" refers to a polypeptide fragment that includes at least one complementarity-determining region (CDR) of an immunoglobulin heavy chain and / or light chain that binds to at least one epitope of the antigen of interest. In this regard, an antigen-binding fragment of an anti-annexin V antibody may include all 1, 2, 3, 4, 5, or 6 CDRs of the variable heavy chain (VH) and variable light chain (VL) sequences derived from an antibody that specifically binds to annexin V. Examples of antigen-binding fragments include portions of full-length antibodies such as Fab, F(ab’)2, Fab’, Fv fragments, minibodies, diabodies, single-domain antibodies (dAbs), single-chain variable fragments (scFvs), and multispecific antibodies formed from antibody fragments, generally proteins that include their antigen-binding regions or variable regions.

[0075] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that, when compared, have a specified percentage of identical nucleotides or amino acid residues. Unless otherwise indicated, percent identity is determined using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST®), available at blast.ncbi.nlm.nih.gov / Blast.cgi, version BLAST+2.13.0.

[0076] II. Apoptosis Apoptosis is a form of programmed cell death that occurs in multicellular organisms. Biochemical events result in characteristic cell changes (morphology) and cell death. These changes include bleb formation, cell shrinkage, nuclear fragmentation, chromatin condensation, DNA fragmentation, and mRNA degradation. The average adult loses billions of cells every day due to apoptosis.

[0077] In contrast to necrosis, which is a form of traumatic cell death resulting from acute cell injury, apoptosis is a highly regulated and controlled process that confers 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 digits undergo apoptosis. Unlike necrosis, apoptosis produces cell fragments called apoptotic bodies, which can be phagocytosed and removed by phagocytes before the cell's contents leak out and cause damage to surrounding cells.

[0078] Apoptosis is a highly regulated process. Apoptosis can be initiated by one of two pathways: the intrinsic and the extrinsic. In the intrinsic pathway, the cell kills itself to sense cell stress, while in the extrinsic pathway, the cell kills itself due to signals from other cells. Weak external signals can also activate the intrinsic pathway of apoptosis. Both pathways induce cell death by activating caspases, which are proteases, or enzymes that break down proteins. Both pathways activate initiator caspases, which then activate executioner caspases, which then kill the cell by indiscriminately breaking down proteins. The cell's membrane undergoes such breakdown, resulting in the release of nucleic acids such as nuclear DNA, mitochondrial DNA, and RNA.

[0079] The intrinsic pathway is also known as the mitochondrial pathway. Mitochondria are essential for multicellular life. Without them, cells would stop respiring aerobically and die rapidly. This fact forms the basis of some apoptosis pathways. Apoptosis proteins that target mitochondria affect mitochondria in various ways. They can cause mitochondrial swelling through the formation of membrane pores, or increase the permeability of the mitochondrial membrane, allowing the leakage of apoptotic effectors.

[0080] During apoptosis, cytochrome c is released from mitochondria by the action of the proteins Bax and Bak. The mechanism of this release is unclear, but it seems to be derived from multiple Bax / Bak homodimers and heterodimers of Bax / Bak inserted into the outer membrane. Once cytochrome c is released, it binds to apoptotic protease-activating factor-1 (Apaf-1) and ATP, which then binds to procaspase-9 to form a protein complex known as the apoptosome. The apoptosome cleaves procaspase to its active form, caspase-9, which then cleaves and activates procaspase to effector caspase-3.

[0081] Mitochondria also release a protein known as SMAC (second mitochondria-derived activator of caspase) into the cytosolic compartment of the cell after an increase in mitochondrial membrane permeability. SMAC binds to proteins that inhibit apoptosis (IAPs), thereby inactivating them and preventing them from halting the process, thus allowing apoptosis to proceed. IAPs also normally suppress the activity of a group of cysteine proteases called caspases that effect cell disassembly. Thus, it can be seen that the actual proteolytic enzymes are indirectly regulated by mitochondrial permeability.

[0082] Two theories for the exogenous direct initiation of the apoptosis mechanism in mammals have been proposed: the TNF-induced (tumor necrosis factor) model and the Fas-Fas ligand-mediated model (both involving receptors of the TNF receptor (TNFR) family that are coupled to exogenous signals).

[0083] TNF-alpha is a cytokine mainly produced by activated macrophages and is a major exogenous mediator of apoptosis. Most cells in the human body have two receptors for TNF-alpha: TNFR1 and TNFR2. Binding of TNF-alpha to TNFR1 has been shown to initiate a pathway leading to caspase activation via the intermediate membrane protein TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD). cIAP1 / 2 can inhibit TNF-alpha 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 through TNFR1 can also induce apoptosis in a caspase-independent manner.

[0084] The Fas receptor (the first apoptosis 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 results in the formation of a death-inducing signaling complex (DISC) containing FADD, caspase-8, and caspase-10. In some types of cells (type I), processed caspase-8 directly activates other members of the caspase family, triggering the execution of apoptosis in the cell. In other types of cells (type II), the Fas-DISC initiates a feedback loop that spirals into an increase in the release of apoptosis-promoting factors from mitochondria and an amplification of caspase-8 activation.

[0085] Defective apoptotic processes are involved in a wide variety of diseases. Excessive apoptosis causes atrophy, while insufficient amounts result in uncontrolled cell growth such as cancer. The progression of human immunodeficiency virus infection to AIDS is mainly due to the depletion of CD4+ T helper lymphocytes in a manner that is too rapid for the body's bone marrow to replenish the cells, impairing the immune system. One mechanism by which T helper cells are depleted is apoptosis resulting from a series of biochemical pathways.

[0086] Inhibition of apoptosis can lead to several cancers, inflammatory diseases, and viral infections. Disruption of the process can result in cells that live beyond their "use-by" date, replicate, and pass on some defective mechanism to their progeny, increasing the likelihood that the cells will become cancerous or diseased. The associated accumulation of cells was originally thought to be due to increased cell proliferation, but is now thought to be due also to decreased cell death. The most common of these diseases is cancer, which is often a disease of excessive cell growth characterized by the overexpression of IAP family members. As a result, malignant cells experience an abnormal response to apoptosis induction: cell cycle regulatory genes (such as p53, ras, or c-myc) are mutated or inactivated in diseased cells, and additional genes (such as bcl-2) also modify their expression in tumors. Some apoptotic factors are essential during mitochondrial respiration (for example, cytochrome C). The pathological inactivation of apoptosis in cancer cells correlates with a frequent respiratory metabolic shift to glycolysis (an observation known as the "Warburg hypothesis").

[0087] Several factors such as Fas receptor and caspases promote apoptosis, while some members of the protein Bcl-2 family inhibit apoptosis. Phosphatidylserine (PS) is a commonly used marker for apoptosis. In normal healthy cells, PS is present on the inner side of the cell membrane. However, during apoptosis, PS translocates to the outer side of the membrane. The association between TNF-alpha and apoptosis indicates the reason why abnormal production of TNF-alpha plays a fundamental role in several human diseases, particularly autoimmune diseases. Apoptosis is known to be one of the major mechanisms of targeted cancer therapy. Luminescent iridium complex-peptide hybrid (IPH) has been designed in recent years, which mimics TRAIL, binds to cell death receptors on cancer cells, and thereby induces their apoptosis.

[0088] Hydrogel beads and compositions and kits containing the same are useful for detecting apoptosis, dead cells, and live cells.

[0089] III. Detection of Apoptosis Using Viability Staining Viability staining is a process of distinguishing live cell populations and dead cell populations within a sample. A viability control is generally a mixture of live and dead cells stained with a discriminatory dye. These dyes are generally classified into one of two broad categories: DNA intercalating dyes and primary amine (protein) dyes. Despite the fact that several DNA-binding dyes are commonly used to identify live cell populations, DNA dye compensation beads are hardly available. Furthermore, existing polystyrene beads used for amine-reactive dyes have fundamentally different properties compared to cell materials and cause difficulties when attempting to work with specific major classes of dyes in important fluorescence channels.

[0090] To create a staining control, apoptosis must be induced in actual cells using a heating method or a chemical method, which is an uneconomical and time-consuming process that is not well standardized. A mixture of live cells and treated cells is stained with a fluorescent annexin V conjugate, which is used to distinguish apoptotic and non-apoptotic populations. In flow cytometry, these controls can be used to set compensation. To eliminate false positives, in addition to annexin V, a live cell-impermeable dye such as propidium iodide or 7AAD is often used. In the later stages of apoptosis, the cell membrane begins to rupture, allowing annexin V to enter the cell and bind to PS on the intracellular portion of the membrane. Live cell-impermeable dyes are used to detect membrane rupture.

[0091] These two dyes are often sold together in an "apoptosis detection" kit. Existing bead products related to annexin V exist, but they are all intended to be used to deplete or select 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 this bead is used to enrich a population of apoptotic cells rather than as a compensation control by itself.

[0092] The ability to identify and measure specific cell types such as apoptotic cells depends on the proper calibration of the measuring instrument. Calibration has relied on the use of purified cells of the cell type of interest. Obtaining these purified cells using a heating method or a chemical method is an uneconomical, time-consuming process that is not well standardized and is prone to batch-to-batch variation. Furthermore, the cells used for calibration can be rare or in short supply. Therefore, there is a need in the art for synthetic compositions having adjustable optical and binding properties that can mimic apoptotic cells.

[0093] Flow cytometry is a technique that enables the rapid separation, counting, and characterization of individual cells, such as potential apoptotic cells, and is routinely used in clinical and experimental settings for a variety of applications. Optics-based flow cytometry relies on directing a light beam at a hydrodynamically focused stream of liquid. Then, several detectors are aimed at the point where the flow passes through the light beam, one in line with the light beam (forward scatter or FSC), and several perpendicular to the light beam (side scatter or SSC). FSC correlates with cell volume, and SSC depends on the internal complexity of the particle (e.g., nuclear shape, amount and type of cytoplasmic granules, or membrane roughness). As a result of these correlations, different specific cell types exhibit different FSC and SSC, enabling the discrimination of cell types.

[0094] Creating a calibration control using the cells of interest is time-consuming, uneconomical, and not sufficiently standardized. Using polystyrene beads as a calibration control results in a control with fundamentally different properties compared to the cell material of interest, creating difficulties when attempting to work with certain major classes of dyes in important fluorescence channels.

[0095] A calibration control for flow cytometry that avoids the problems associated with the use of cells of interest is provided herein. The calibration controls described herein include hydrogel beads.

[0096] IV. Hydrogel Beads (a) Hydrogel beads are provided herein that include a polymerizing monomer and a bifunctional monomer, and in some embodiments (b) a pre-apoptotic signal binder. Also provided herein are hydrogel beads that include (a) a polymerizing monomer, and in some embodiments a bifunctional monomer, and (b) a pre-apoptotic signal. In embodiments, the hydrogel beads include (c) an encapsulated nucleic acid. In embodiments, the hydrogel beads include (d) additional components.

[0097] IV-A. Overlapping Monomers and Bifunctional Monomers In embodiments, the polymerizing monomers and bifunctional monomers of the hydrogel beads described herein form a hydrogel. A "hydrogel" is a material that swells in the presence of water (i.e., "wet state") and shrinks in the absence of water (or by reducing the amount of water) (i.e., "dehydrated state") but does not dissolve in water, and includes a polymeric three-dimensional network. As used herein, the term "hydrogel" refers to the material in its wet or dehydrated state. Swelling or water absorption is the result of the presence of hydrophilic functional groups attached to or dispersed within the polymeric network. Crosslinking between adjacent polymers results in the water-insolubility of these hydrogels. Crosslinking can be due to chemical (i.e., covalent) or physical (i.e., van der Waals forces, hydrogen bonds, ionic forces, etc.) bonds. These chemical crosslinks can also be hydrolyzed under certain conditions, reversing the insolubility of the hydrogel. Multiple 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.

[0098] In some embodiments, the hydrogel contains, by weight, more than about 30%, more than about 40%, more than about 50%, more than about 55%, more than about 60%, more than about 65%, more than about 70%, more than about 75%, more than about 80%, more than about 85%, more than about 90% or more than about 95% water. In embodiments, the hydrogel has a water content of about 10 weight percent to about 95 weight percent, or about 20 weight percent to about 95 weight percent, or about 30 weight percent to about 95 weight percent, or about 40 weight percent to about 95 weight percent, or about 50 weight percent to about 95 weight percent, or about 60 weight percent to about 95 weight percent, or about 70 weight percent to about 95 weight percent, or about 80 weight percent to about 95 weight percent.

[0099] In embodiments, the hydrogel retains the same shape under dehydrated conditions and wetted conditions. For example, if the hydrogel has a substantially spherical shape under dehydrated conditions, the hydrogel will be substantially spherical under wetted conditions.

[0100] In embodiments, the hydrogel forms particles. In embodiments, the hydrogel is synthesized by polymerizing one or more of the 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 radical formation and reaction progression induced by ultraviolet light. In embodiments, acrylate is the monomer to be polymerized. In embodiments, acrylamide is the monomer to be polymerized.

[0101] In an embodiment, the acrylamide is a polymerizable carbohydrate-derivatized acrylamide as described in U.S. Patent No. 6,107,365, the disclosure of which is incorporated by reference in its entirety for all purposes. As described herein and known to those of skill in the art, the specific attachment of acrylamide groups to sugars readily accommodates a series of monosaccharides and higher-order polysaccharides, such as synthetic or naturally sourced polysaccharides, such as glycoproteins found in serum or tissue.

[0102] In an embodiment, an acrylate-functionalized poly(ethylene) glycol monomer is polymerized. In an embodiment, an acrylamide-functionalized PEG is polymerized.

[0103] In an embodiment, the hydrogel beads comprise a monofunctional monomer polymerized with at least one bifunctional monomer. In an embodiment, the hydrogel beads comprise a polymer of acrylamide (i.e., polyacrylamide) and bis-acrylamide (a bifunctional monomer).

[0104] In embodiments, the hydrogel beads provided herein include a difunctional monomer polymerized with a second difunctional monomer. In embodiments, the hydrogel beads including a difunctional monomer polymerized with a second difunctional monomer include polymers having a mixed composition including compatibility chemistries such as acrylamide, bis-acrylamide, and bis-acrylamide structure homologs that include a wide range of additional chemistries. In embodiments, one functional group of the difunctional monomer is an alkyne and the other functional group is an ester, amide, ketone, aldehyde, azide, alkene, alcohol, amine, or carboxylic acid. In embodiments, one functional group of the difunctional monomer is an alkyne and the other functional group is an alcohol, amine, or carboxylic acid. In embodiments, one functional group of the difunctional monomer is an alkene and the other functional group is an amine. In embodiments, one functional group of the difunctional monomer is an alkene and the other functional group is an alcohol, amine, or carboxylic acid. In embodiments, one functional group of the difunctional monomer is an alkene and the other functional group is an ester, amide, ketone, aldehyde, azide, alkene, alcohol, amine, or carboxylic acid.

[0105] In an embodiment, the hydrogel beads provided herein include a polymerizable monofunctional monomer and are monofunctional acrylic monomers. Non-limiting examples of monofunctional acrylic monomers for use herein are acrylamide, methacrylamide; N-alkylacrylamides such as N-ethylacrylamide, N-isopropylacrylamide or N-tert-butylacrylamide; N-alkylmethacrylamides such as N-ethylmethacrylamide or N-isopropylmethacrylamide; N,N-dialkylacrylamides such as N,N-dimethylacrylamide and N,N-diethylacrylamide; N-[(dialkylamino)alkyl]acrylamides such as N-[3-(dimethylamino)propyl]acrylamide or N-[3-(diethylamino)propyl]acrylamide; N-[(dialkylamino)alkyl]methacrylamides 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.

[0106] In an embodiment, the bifunctional monomer is selected from the group consisting of allylamine, allyl alcohol, allyl isothiocyanate, allyl chloride and allyl maleimide.

[0107] In an embodiment, the bifunctional monomer is a bifunctional acrylic monomer. Non-limiting examples of bifunctional acrylic monomers are N,N'-methylenebisacrylamide, N,N'-methylenebismethacrylamide, N,N'-ethylenebisacrylamide, N,N'-ethylenebismethacrylamide, N,N'-bisacryloylcystamine, N,N'-propylenebisacrylamide and N,N'-(1,2-dihydroxyethylene)bisacrylamide.

[0108] As described in U.S. Patent No. 6,657,030, which is hereby incorporated by reference in its entirety for all purposes, higher order branched and linear comonomers can be substituted in a polymer mixture to adjust the refractive index while maintaining the polymer density.

[0109] In one embodiment, the biomonomer is functionalized with acrylamide or acrylate. For example, in one embodiment, a polymerizable acrylamide-functionalized biomolecule is an acrylamide- or acrylate-functionalized protein (e.g., acrylamide-functionalized collagen or a functionalized collagen domain), an acrylamide- or acrylate-functionalized peptide, or an acrylamide- or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide.

[0110] Any monosaccharide, disaccharide, or polysaccharide (functionalized or otherwise) can be used as a hydrogel monomer. In one embodiment, an acrylamide- or acrylate-functionalized monosaccharide, disaccharide, or polysaccharide 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 yet another embodiment, a glycosaminoglycan (GAG) is used as a polymerizable monomer in the hydrogels provided herein. In a further embodiment, the GAG is chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate, or hyaluronic acid (also referred to in the art as hyaluron or hyaluronate) and is used as a polymerizable hydrogel monomer. The additional scope of compatible biomonomers and their reactive chemistries are known to those of skill in the art and follow general principles of chemical reactivity.

[0111] Examples of biocompatible monomers for use with the hydrogels described herein include, in one embodiment, ethylene glycol dimethacrylate (EGDMA), 2-hydroxyethyl methacrylate (HEMA), methyl methacrylate (MMA), methacryloxymethyltrimethylsilane (TMS-MA), N-vinyl-2-pyrrolidone (N-VP), styrene, or combinations thereof.

[0112] Examples of naturally occurring hydrogels useful in the present disclosure include various polysaccharides obtainable from natural sources such as plants, algae, fungi, yeast, marine invertebrates, and arthropods. Non-limiting examples include agarose, dextran, chitin, cellulose-based compounds, starch, derivatized starch, and the like. These generally have repeating glucose units as a major part of the polysaccharide backbone. The crosslinking chemistry of such polysaccharides is known in the art; see, for example, 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).

[0113] In one embodiment, hyaluronan is used as a hydrogel monomer (either as a single monomer or as a comonomer). In one embodiment, hyaluronan is functionalized, for example, with acrylate or acrylamide. Hyaluronan is a high molecular weight GAG composed of disaccharide repeat units of N-acetylglucosamine and glucuronic acid linked together via alternating β-1,4 and β-1,3 glycosidic bonds. In the human body, hyaluronate is found in several soft connective tissues including the skin, umbilical cord, synovial fluid, and vitreous humor. Thus, in one embodiment, it is desirable to mimic the optical properties of one or more of skin cells, umbilical cord cells, or vitreous humor cells. In one embodiment, hyaluronan is used as a hydrogel monomer. Methods for making hydrogel particles are described in Xu et al., (2012). Soft Matter. 8, pp. 3280-3294, the disclosure of which is incorporated herein in its entirety for all purposes. As described herein, hyaluronan can be derivatized with various reactive handles depending on the desired crosslinking chemistry and other monomers used to form the hydrogel particles.

[0114] 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).

[0115] Other polysaccharides for use as hydrogel monomers or comonomers include, but are not limited to, agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, carrageenan polysaccharides (e.g., e.g., kappa, iota or lambda classes), cellodextrin, cellulins, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucorunoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, pleuran, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or combinations thereof. As described throughout, the polysaccharides can be further functionalized depending on the desired cross-linking chemistry and / or additional comonomers used in the hydrogel. For example, in one embodiment, one or more of the polysaccharides described herein are functionalized with acrylate or acrylamide.

[0116] In one embodiment, the individual hydrogel particles or a plurality thereof include, as hydrogel monomers or a plurality thereof, peptides, proteins, protein domains, or combinations thereof. In a further embodiment, the protein is a structural protein or a domain thereof, such as silk, elastin, titin or collagen, or a domain thereof. In one embodiment, the protein is an extracellular matrix (ECM) component (e.g., collagen, elastin, proteoglycan). In still further embodiments, the structural protein is collagen. In still further embodiments, the collagen is type I collagen, type II collagen or type III collagen or a combination thereof. In another embodiment, the hydrogel monomer includes a proteoglycan. In a further embodiment, the proteoglycan is decorin, biglycan, testican, brevican, fibromodulin, lumican, or a domain thereof.

[0117] In another embodiment, an acrylate-functionalized structural protein hydrogel monomer is used as a component of the hydrogels provided herein (e.g., an acrylate-functionalized protein or protein domain, such as silk, elastin, titin, collagen, proteoglycan, or a functionalized domain thereof). In a further embodiment, the acrylate-functionalized structural protein hydrogel monomer includes a proteoglycan, such as decorin, biglycan, testican, brevican, fibromodulin, lumican, or a domain thereof.

[0118] In one embodiment, the PEG monomer and oligopeptide can mimic extracellular matrix proteins used in the hydrogels provided herein, such as, for example, vinylsulfone-functionalized multi-arm PEG, integrin-binding peptides, and bis-cysteine matrix metalloproteinase peptides as described by Lutolf et al., (2003). Proc. Natl. Acad. Sci. U.S.A. 100, 5413-5418 (incorporated by reference in its entirety for all purposes). In this particular embodiment, the hydrogel is formed by a Michael-type addition reaction between a dithiolated oligopeptide on the PEG and a vinylsulfone group. The scope of additional compatible chemistries that can be incorporated herein will be apparent to those skilled in the art and follows general principles of chemical reactivity (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).

[0119] Other bioactive domains in native proteins can also be used as hydrogel monomers or a part thereof. For example, cell adhesive integrin-binding domains, controlled release affinity binding domains, or transglutaminase crosslinking domains can be used in the hydrogels provided herein. Details for generating such hydrogels can be found in 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.

[0120] In one embodiment, recombinant DNA methods are used to create proteins designed to gel in response to changes in pH or temperature, for example, by the methods described by Petka et al., (1998). Science 281, pp. 389-392 (incorporated herein by reference in its entirety for all purposes). Briefly, the protein consists of a terminal leucine zipper domain adjacent to a water-soluble polyelectrolyte segment. In a nearly neutral aqueous solution, the coiled-coil aggregates of the terminal domains form a three-dimensional hydrogel polymer network.

[0121] Additional ranges of biocompatible monomers that can 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 (incorporated herein by reference in its entirety for all purposes). Other monomers are provided in 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 programmes, ISBN 91-631-4985-0, the disclosures of each of which are incorporated herein by reference in their entirety.

[0122] In an embodiment, the polymerization monomer is a homopolymer. The term "homopolymer" refers to a polymerization monomer containing a single repeating monomer unit. In an embodiment, the polymerization monomer is a copolymer. The term "copolymer" refers to a polymer made by the reaction of two different monomers. The two different monomers are called 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 of the comonomers are difunctional monomers.

[0123] In an embodiment, the hydrogel is synthesized in the presence of a crosslinking agent. In an embodiment, the crosslinking agent is selected from any one of ethylene glycol dimethacrylate (EGDMA), tetraethylene glycol dimethacrylate, and N,N'-methylenebisacrylamide.

[0124] In an embodiment, the hydrogel is synthesized in the presence of a polymerization initiator. In an embodiment, the polymerization initiator is a persulfate or an equivalent initiator that catalyzes radical formation. In an embodiment, the persulfate is any water-soluble persulfate. Non-limiting examples of water-soluble persulfates include ammonium persulfate and alkali metal persulfates. In an embodiment, the alkali metal persulfate is an alkali metal persulfate (lithium, sodium, or potassium). In an embodiment, the persulfate is ammonium persulfate or potassium persulfate. In a further embodiment, the polymerization of the hydrogel provided herein is initiated by ammonium persulfate.

[0125] In an embodiment, the polymerization of the hydrogel is accelerated by an accelerator that can catalyze the formation of chemical side groups that are unstable to polymerization. In an embodiment, the accelerator is a tertiary amine. In an embodiment, the tertiary amine is a water-soluble tertiary amine. In an embodiment, the accelerator is N,N,N’,N’-tetramethylethylenediamine, 3-(dimethylamino)propionitrile or N,N,N’,N’-tetramethylethylenediamine (TEMED). In an embodiment, the accelerator is 2,2’-azobis(isobutyronitrile) (AIBN).

[0126] In an embodiment, the amount of the monomer can be varied, for example, to obtain certain optical or morphological properties that are substantially the same as the optical or morphological properties of the target cells. In an embodiment, the monomer is present in about 10 wt% to about 95 wt% of the hydrogel. In an embodiment, the monomer is present in the hydrogel by weight at 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 91%, about 92%, about 93%, about 94%, about 95% (including all values and sub-ranges therebetween including both ends).

[0127] In an embodiment, the polymerized monomer is present at about 10 weight percent to about 95 weight percent of the hydrogel. In an embodiment, the polymerized monomer is present at about 15 weight percent to about 95 weight percent of the hydrogel. In an embodiment, the polymerized monomer is present at about 20 weight percent to about 95 weight percent of the hydrogel. In an embodiment, the polymerized monomer is present, by weight, at 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 91%, about 92%, about 93%, about 94%, about 95% (including all values and subranges therebetween, including the endpoints).

[0128] In an embodiment, the difunctional monomer is present at about 10 weight percent to about 95 weight percent of the hydrogel. In an embodiment, the difunctional monomer is present at about 15 weight percent to about 95 weight percent of the hydrogel. In an embodiment, the difunctional monomer is present at about 20 weight percent to about 95 weight percent of the hydrogel. In an embodiment, the difunctional monomer is present by weight at 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 91%, about 92%, about 93%, about 94%, about 95% (including all values and subranges therebetween including the ends).

[0129] In embodiments, the comonomer is present in about 10 weight percent to about 95 weight percent of the hydrogel. In embodiments, the comonomer is present in about 15 weight percent to about 95 weight percent of the hydrogel. In embodiments, the comonomer is present in about 20 weight percent to about 95 weight percent of the hydrogel. In embodiments, the comonomer is present in 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 91%, about 92%, about 93%, about 94%, about 95% (including all values and subranges therebetween including the endpoints) by weight of the hydrogel.

[0130] In some embodiments, the monomer is selected from any one of 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, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate.

[0131] In an embodiment, the monomer is selected from any one of lactic acid, glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, their derivatized versions, or a combination thereof.

[0132] In the embodiment, the monomers are 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-diphenylmethylacrylamide, N-(4-methylphenyl)methylacrylamide, N-1-naphthylacrylamide, N-4-nitrophenylacrylamide, N-(2-phenylethyl)acrylamide, N-triphenylmethylacrylamide, N-(4-hydroxyphenyl)acrylamide, N,N-methylphenylacrylamide, N,N-phenylphenylethylacrylamide, N-diphenylmethylmethacrylamide, N-(4-methylphenyl)methylmethacrylamide, N-1-naphthylmethacrylamide, N-4-nitrophenylmethacrylamide, N-(2-phenylethyl)methacrylamide, N-triphenylmethylmethacrylamide, N-(4-hydroxyphenyl)methacrylamide, N,N-methylphenylmethacrylamide, N,It is selected from any one of N'-phenylphenyl ethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine or 2-vinylpyridine.,

[0133] In an embodiment, the monomer is selected from any of the monomers disclosed in U.S. Patent No. 6,657,030, which is hereby incorporated by reference in its entirety for all purposes.

[0134] In an embodiment, the monomer is a synthetic monomer. In an embodiment, the monomer is a biomonomer.

[0135] In an embodiment, the monomer is selected from any one 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 hereby incorporated by reference in its entirety for all purposes.

[0136] In embodiments, the hydrogel is a synthetic hydrogel. The synthetic hydrogel contains synthetic monomers. In embodiments, the hydrogel is a biogel. The biogel contains biomolecules such as peptides, proteins, monosaccharides, disaccharides, polysaccharides or carbohydrates. In embodiments, the biogel contains 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 contains synthetic components as well as biomolecules and / or functional groups found on biomolecules. In embodiments, proteins, peptides or carbohydrates can be used as individual monomers to form hydrogels, with or without synthetic monomers (or polymers), in combination with chemically compatible comonomers and crosslinking chemistries. Suitable crosslinking chemistries include, but are not limited to, amines, carboxyls, and other reactive chemical side groups.

[0137] In embodiments, one or more of the monomer, comonomer, difunctional monomer is bis / acrylamide at various crosslinking ratios. In embodiments, one or more of the monomer, comonomer, difunctional monomer contains allylamine. In embodiments, one or more of the monomer, comonomer, difunctional monomer provides chemical functionality for secondary labeling / conjugation. In embodiments, one or more of the monomer, comonomer, difunctional monomer contains alginate. For example, the hydrogels of the present disclosure can be constructed using hydrazine (e.g., together with an NHS ester compound) or an EDC coupling reaction (e.g., together with a maleimide compound).

[0138] In an embodiment, the polymerization monomer is a biodegradable monomer. In an embodiment, the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. In some embodiments, the biodegradable monomer is a structural polysaccharide. In an embodiment, the biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, alguronate, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, cellulase, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage gluxan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, and combinations thereof.

[0139] In an embodiment, the hydrogel is biodegradable. In an embodiment, the hydrogel comprises a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. In an embodiment, the hydrogel comprises a structural polysaccharide. In an embodiment, the hydrogel comprises agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, cellulins, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isomalodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

[0140] In an embodiment, the hydrogel beads contain a biodegradable polymer as a monomer. In an embodiment, the biodegradable polymer is a poly(ester) based on polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA), and copolymers thereof. These polymers can be decomposed by hydrolysis and dissolve the polymer particles. In an embodiment, the biodegradable polymer is a carbohydrate or a protein, or a combination thereof. In one embodiment, monosaccharides, disaccharides or polysaccharides (e.g., glucose, sucrose or maltodextrin), peptides, proteins (or domains thereof) are used as hydrogel monomers. In an embodiment, the biodegradable polymer is a poly(hydroxyalkanoate) of the PHB-PHV class, additional poly(esters), or natural polymers, such as modified poly(saccharides), such as starch, cellulose, and chitosan. In an embodiment, the biocompatible polymer is an adhesive protein, cellulose, carbohydrate, starch (e.g., maltodextrin, 2-hydroxyethyl starch, alginic acid), dextran, lignin, polyamino acid, amino acid or chitin. Such biodegradable polymers are commercially available, for example, from Sigma Aldrich (St. Louis, Missouri).

[0141] In embodiments, the protein monomer contains only natural amino acids. In embodiments, the protein monomer contains non-natural amino acids. For example, non-natural amino acids (e.g., those incorporated into non-ribosomal peptides or synthetically introduced via a synthetic approach (e.g., 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 self-assembling artificial proteins and proteins having such protein domains can also be used as hydrogel monomers. The range of non-natural (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, the disclosure of which is incorporated by reference in its entirety for all purposes). In embodiments, the biodegradable polymer is used as a comonomer. In embodiments, the biodegradable polymer in one embodiment is a bifunctional monomer.

[0142] In embodiments, the hydrogel beads described herein contain a degradable polymer as a monomer. In embodiments, the degradable polymer is a poly(ester) based on PLA, PGA, PCL, PLGA, and their copolymers. In embodiments, the degradable polymer is based on any one of the monomers described herein and can be degradable 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 can be degraded by exposure to potassium persulfate.

[0143] In embodiments, the degradation of individual hydrogel particles or a plurality thereof, whether by biodegradation means, dissolution, or otherwise, can result in the release of the substances contained therein. For example, when the substance is a biomolecule such as hemoglobin, the degradation of the hydrogel particles containing hemoglobin can enable the measurement of hemoglobin separated from the hydrogel monomers. In another example, when the substance is a biomolecule such as hemoglobin, the degradation of the hydrogel particles containing hemoglobin can be by a lysis buffer.

[0144] In embodiments, the refractive index of the hydrogel may be varied by adjusting the concentration of the bifunctional monomer and / or the polymerizing monomer.

[0145] In embodiments, the hydrogel beads described herein can be dissolved. The dissolvable synthetic beads of the present invention enable the user to measure both intact beads and dissolved beads. In embodiments, the hydrogel beads are dissolved in a lysis buffer. In embodiments, the lysis buffer is ammonium chloride. Hematological lysis buffers often use ammonium chloride, including Thermo Fisher Scientific's 1× RBC lysis buffer and 10× RBC lysis buffer. Hematological lysis buffers used in clinical blood samples are designed to lyse non-nucleated red blood cells and preserve white blood cells for the quantitative measurement of white blood cell count and hemoglobin. Other lysis buffers can be designed to dissolve engineered particles containing strong reducing agents such as dithiothreitol (DTT) or β-mercaptoethanol (BME). Additional non-limiting examples include divalent ions such as ethylenediaminetetraacetic acid (EDTA) or citrate.

[0146] In an embodiment, the hydrogel beads are formed by precipitation polymerization as described in Elbert (2011), Acta Biomater. 7, pp. 31 - 56, which is hereby incorporated by reference in its entirety for all purposes. Precipitation polymerization is a technique for producing microparticles by taking advantage of the difference in solubility between monomers and polymers. Specifically, large polymer chains are generally known to have lower solubility than small polymer chains. Thus, phase separation can be promoted when a specific molecular weight is exceeded. Precipitation polymerization begins initially as solution polymerization in a single - phase homogeneous system. Immediately after the start of polymerization, in one embodiment, a relatively high concentration of polymer chains is present, promoting phase separation by nucleation. As the polymerization progresses, the concentration of polymer chains decreases, and existing particles capture the chains before new particle nucleation can occur. Thus, particle nucleation occurs only for a short time immediately after the start of the reaction and, in one embodiment, results in a narrow particle size distribution. In an embodiment, the hydrogel beads can be formed by one or more methods selected from the group consisting of lithographic particle formation (Helgeson et al., (2011). Curr. Opin. Colloid. Interface Sci. 16, pp. 106 - 117, which is hereby incorporated by reference in its entirety for all purposes), membrane emulsification (e.g., by the method of the microsieve emulsification technique described by Nanomi B.V. (Netherlands)), microchannel emulsification (Sugiura et al., (2002). Languimir 18, pp. 5708 - 5712, which is hereby incorporated by reference in its entirety), or bulk emulsification (available at snf.com.au / downloads / Emulsion_Handbook_E.pdf from SNF Floerger, which is hereby incorporated by reference in its entirety).

[0147] In an embodiment, the hydrogel beads are formed in a microfluidic device having two oil channels that focus on the central flow of the aqueous monomer solution. In an embodiment, the droplets are formed at the interface of the two channels and the central flow to separate the droplets in the water-in-oil emulsion. In an embodiment, after the droplets are formed, they are stabilized prior to polymerization. In an embodiment, the droplets are stabilized by adding a surfactant to the oil phase. In an embodiment, the droplets are not stabilized prior to polymerization. In an embodiment, the polymerization of the monomer is caused by adding an accelerator (e.g., N,N,N’,N’-tetramethylethylenediamine) to one or both of the oil channels after the first droplets are formed.

[0148] In an embodiment, the aqueous monomer solution provided above can contain a single monomer species or a plurality of monomer species. In an embodiment, the aqueous monomer solution contains a comonomer, a bifunctional monomer, or a combination thereof. In an embodiment, the monomer or monomers include a bifunctional monomer. In an embodiment, the monomer is one of the above monomers. In an embodiment, a comonomer is used to adjust the forward scattering or the side scattering. In an embodiment, a comonomer is used to adjust the refractive index of the hydrogel beads.

[0149] In an embodiment, the central flow of the aqueous monomer solution contains a crosslinking agent. In an embodiment, the crosslinking agent is N,N’-bisacrylamide. In an embodiment, the central flow of the aqueous monomer solution contains a crosslinking agent, an accelerator, and a monomer. In an embodiment, the aqueous monomer solution contains an initiator. In an embodiment, the initiator is an oxidizing agent. In an embodiment, the oxidizing agent is ammonium persulfate.

[0150] In an embodiment, the hydrogel beads are produced by polymerizing droplets. Microfluidic methods for generating a plurality of droplets including fluid droplets and hardened droplets are described in U.S. Patent Application Publication No. 2011 / 0218123 and U.S. Patent No. 7,294,503, each of which is hereby incorporated 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, where the first fluid and the second fluid are substantially immiscible (e.g., droplets containing an aqueous liquid are substantially surrounded by an oil-based liquid).

[0151] IV-B. Pre-apoptosis Signals and Pre-apoptosis Signal Binders In an embodiment, the hydrogel beads described herein include a pre-apoptosis signal or a pre-apoptosis signal binder. In an embodiment, the pre-apoptosis signal binder is present on a cell undergoing apoptosis and binds to the pre-apoptosis signal. In an embodiment, the hydrogel beads containing the pre-apoptosis signal binder serve as a mimic of apoptotic cells. In some embodiments, the pre-apoptosis signal binder is artificially designed to bind to a known pre-apoptosis signal (e.g., an antibody targeting a pre-apoptosis signal).

[0152] Table 1A includes pre-apoptosis signal binders and the pre-apoptosis signals to which they bind. In an embodiment, the hydrogel beads described herein include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different pre-apoptosis signal binders, including all ranges and sub-ranges therebetween. In an embodiment, the hydrogel beads described herein include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different pre-apoptosis signals, including all ranges and sub-ranges therebetween.

Table 1A-1

Table 1A-2

[0153] The following patent documents and publications describe pre-apoptotic signal binders and pre-apoptotic signals and are hereby incorporated by reference in their entirety for all purposes: Elmore, Toxicol Pathol. 2007;35(4):495-516; Armstrong et al., EMBO Rep. April 1, 2011;12(4):287-288; and U.S. Patent Application Publication No. 2022 / 0143160.

[0154] In an embodiment, the pre-apoptosis 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 to annexin V (UniProt accession number 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 the 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 (e.g., domain V) (UniProt accession number D9IWP9), 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 number 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: 102, 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 number 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 the LDL receptor-related protein (UniProt accession number P01130), or at least 75% to an anti-calreticulin antibody or an antigen-binding fragment thereof,It comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity.

[0155] In an embodiment, the pre-apoptosis signal includes annexin V. Annexin V binds to phosphatidylserine present on the outer leaflet of the plasma membrane of apoptotic cells. In an embodiment, annexin V is from human, rabbit, mouse, Ailuropoda melanoleuca, Aotus nancymaae, Balaenoptera acutorostrata scammoni, Balaenoptera musculus, Bos indicus, Bos taurus, Bos indicus, Bos mutus, Bos taurus, Bubalus bubalis, Callithrix jacchus, Camelus bactrianus, Canis lupus familiaris, Capra hircus, Carlito syrichta, Castor canadensis, bovine, Cebus imitator, Cervus canadensis, Cervus elaphus, Cervus hanglu yarkandensis, Delphinapterus leucas, Dipodomys ordii, Dipodomys spectabilis, Elephas maximus indicus, Equus przewalskii, Eschrichtius robustus, Felis catus, Gorilla gorilla, Gorilla beringer, Gulo gulo luscus, Halichoerus grypus, Hyaena hyaena, Hylobates moloch, Ictidomys tridecemlineatus, Jaculus jaculus, Lagenorhynchus obliquidens, Lemur catta, Lipotes vexillifer, Loxodonta africana, Macaca fascicularis, Macaca mulatta, Mandrillus leucophaeus, Marmota flaviventris, Marmota marmota marmota, Marmota monax, Moschus berezovskii, Muntiacus muntjak, Mustela putorius furo, NeogaleIt is derived from a species selected from the group consisting of vison, Neomonachus schauinslandi, Nomascus leucogenys, Nyctereutes procyonoides, Odobenus rosmarus divergen, Odocoileus virginianus texanus, Orcinus orca, Ovis aries, Pan troglodytes, Papio anubis, Perognathus longimembris pacificus, Phoca vitulina, Physeter catodon, Piliocolobus tephrosceles, Propithecus coquereli, Rangifer tarandus platyrhyncus, Rhinopithecus bieti, Saimiri boliviensis boliviensis, Sciurus carolinensis, Sorex araneus, Sus scrofa, Trachypithecus francoisi, Tupaia chinensis, Tursiops truncatus, Urocitellus parryii, Ursus maritimus, Vulpes lagopus, and Zalophus californianus. In an embodiment, the annexin V protein 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 with an annexin V protein having any one of the amino acid sequences of SEQ ID NOs: 1 to 101.

[0156] In embodiments, the pre-apoptosis signal comprises the polypeptide of SEQ ID NO: 103. In embodiments, the polypeptide of SEQ ID NO: 103 is cyclic. In embodiments, the pre-apoptosis signal comprises the apo-15 peptide. Apo-15 is a cyclic peptide having the amino acid sequence GRKKWFW (SEQ ID NO: 104). The structure of the apo-15 peptide is described in Barth et al., Nat Commun. Aug 12, 2020;11(1):4027, which is hereby incorporated by reference in its entirety for all purposes. In embodiments, the cyclic apo-15 peptide comprises any of the fluorescent dyes 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 hereby incorporated by reference in its entirety for all purposes.

[0157] In embodiments, the pre-apoptosis signal is a phospholipid-binding protein. In embodiments, the phospholipid-binding protein is β2-glycoprotein 1 (β2GPI) or prothrombin. Both of these proteins bind to phosphatidylserine on apoptotic cells. In embodiments, the pre-apoptosis signal is domain V of β2GPI. β2GPI and prothrombin are described in McDonnell et al., Blood Rev. Jan 2020;39:100610 and Houston et al., Mol Cell Biochem. Feb 2011;348(1-2):109-15. These references are hereby incorporated by reference in their entirety for all purposes.

[0158] In an embodiment, the pre-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 hereby incorporated by reference in their entirety for all purposes: Lauber et al., Cell Death Differ. September 2013;20(9):1230-40 and Borishenko et al., Cell Death Differ. August 2004;11(8):943-5.

[0159] In an embodiment, the pre-apoptotic signal is a phosphatidylserine receptor or a fragment thereof. Phosphatidylserine receptors are expressed by phagocytes. There are multiple phosphatidylserine receptors with different structures, cell-type expression, and the ability to bind phosphatidylserine. The following reference describes phosphatidylserine receptors and is hereby incorporated by reference in its entirety for all purposes: Naeini et al., Cell Mol Biol Lett. March 26, 2020;25:23. In an embodiment, the phosphatidylserine receptor is selected from any one of 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 includes the UniProt accession numbers of the aforementioned phosphatidylserine receptors. In an embodiment, the pre-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 to any one of the phosphatidylserine receptors in Table 1B.

Table 1B

[0160] In an embodiment, the pre-apoptosis signal is CD36. CD36 binds to thrombospondin-1 expressed on apoptotic cells. The following paper describes the role of CD36 in apoptosis and is hereby incorporated by reference in its entirety for all purposes: Fadok et al., J Immunol. December 1, 1998; 161(11): 6250-7.

[0161] In an embodiment, the pre-apoptosis signal includes the LDL receptor-related protein. The LDL receptor-related protein binds to calreticulin and initiates clearance of apoptotic cells. The following paper describes the role of the LDL receptor-related protein in apoptosis and is hereby incorporated by reference in its entirety: Gardai et al., Cell. October 21, 2005: 123(2): 321-324. In an embodiment, the pre-apoptosis signal is an anti-calreticulin antibody or an antigen-binding fragment thereof.

[0162] In an embodiment, any of the pre-apoptosis signals described herein may include a fluorescent dye. In an embodiment, the fluorescent dye is any of the fluorescent dyes described herein.

[0163] In an embodiment, the pre-apoptosis signal binder is selected from phosphatidylserine, an anti-annexin V antibody or an antigen-binding fragment thereof, annexin I, calreticulin, an anti-CD36 antibody or an antigen-binding fragment thereof, thrombospondin-1 (TSP-1), an anti-β2-glycoprotein I antibody or an antigen-binding fragment thereof, an anti-milk fat globule-EGF-factor 8 (MFG-E8) or an antigen-binding fragment thereof, an anti-phosphatidylserine receptor or an antigen-binding fragment thereof, or an anti-LDL receptor-related protein or an antigen-binding fragment thereof.

[0164] In an embodiment, the pre-apoptosis signal binder 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 having 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 to any one of SEQ ID NOs: 1-101. In an embodiment, the anti-annexin V antibody or an antigen-binding fragment thereof is from a human, rabbit, mouse, Ailuropoda melanoleuca, Aotus nancymaae, Balaenoptera acutorostrata scammoni, Balaenoptera musculus, Bos indicus × Bos taurus, Bos indicus, Bos mutus, Bos taurus, Bubalus bubalis, Callithrix jacchus, Camelus bactrianus, Canis lupus familiaris, Capra hircus, Carlito syrichta, Castor canadensis, bovine, Cebus imitator, Cervus canadensis, Cervus elaphus, Cervus hanglu yarkandensis, Delphinapterus leucas, Dipodomys ordii, Dipodomys spectabilis, Elephas maximus indicus, Equus przewalskii, Eschrichtius robustus, Felis catus, Gorilla gorilla, Gorilla beringer, Gulo gulo luscus, Halichoerus grypus, human (HomoIt binds to annexin V derived from a species selected from any one of Homo sapiens, Hyaena hyaena, Hylobates moloch, Ictidomys tridecemlineatus, Jaculus jaculus, Lagenorhynchus obliquidens, Lemur catta, Lipotes vexillifer, Loxodonta africana, Macaca fascicularis, Macaca mulatta, Mandrillus leucophaeus, Marmota flaviventris, Marmota marmota marmota, Marmota monax, Moschus berezovskii, Muntiacus muntjak, Mustela putorius furo, Neogale vison, Neomonachus schauinslandi, Nomascus leucogenys, Nyctereutes procyonoides, Odobenus rosmarus divergen, Odocoileus virginianus texanus, Orcinus orca, Ovis aries, Pan troglodytes, Papio anubis, Perognathus longimembris pacificus, Phoca vitulina, Physeter catodon, Piliocolobus tephrosceles, Propithecus coquereli, Rangifer tarandus platyrhyncus, Rhinopithecus bieti, Saimiri boliviensis boliviensis, Sciurus carolinensis, Sorex araneus, Sus scrofa, Trachypithecus francoisi, Tupaia chinensis, Tursiops truncatus, Urocitellus parryii, Ursus maritimus, Vulpes lagopus, and Zalophus californianus.

[0165] In an embodiment, the pre-apoptotic signal binder is phosphatidylserine. The structure of phosphatidylserine includes a glycerol backbone linked 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. [Chemical formula]

[0166] In an embodiment, the pre-apoptotic signal binder is annexin I. Annexin I is a glucocorticoid-regulated protein involved in phagocytosis, cell signaling, and regulation of proliferation, and is hypothesized to be a mediator of the glucocorticoid action in the control of inflammation and anterior pituitary hormone release. Annexin I expression is increased in apoptotic cells, cross-links phosphatidylserine on apoptotic cells to phagocytes, and seems to play a role in enhancing the recognition of apoptotic cells by phagocytes such as macrophages.

[0167] In an embodiment, any one of the pre-apoptotic signal binders described herein may include a fluorescent dye. In an embodiment, the fluorescent dye is any fluorescent dye described herein.

[0168] In some embodiments, the pre-apoptotic signal binder or pre-apoptotic signal is embedded within a hydrogel matrix. In some embodiments, the pre-apoptotic signal binder or pre-apoptotic signal is attached to the surface of the hydrogel. In some embodiments, the pre-apoptotic signal binder or pre-apoptotic signal is attached to the hydrogel via a functional group (e.g., an amine group or biotin or streptavidin).

[0169] IV-C. Encapsulated Nucleic Acid In an embodiment, the hydrogel beads described herein further contain encapsulated nucleic acid. The hydrogel beads containing the encapsulated nucleic acid serve as a mimic of dead cells. Dead cells typically have a damaged cell membrane and thus have exposed DNA.

[0170] In an embodiment, the hydrogel beads described herein contain (a) a polymerizable monomer and a bifunctional monomer, (b) a pre-apoptotic signal, and (c) an encapsulated nucleic acid.

[0171] In an embodiment, the hydrogel beads described herein contain (a) a polymerizable monomer and a bifunctional monomer, (b) a pre-apoptotic signal binder, and (c) an encapsulated nucleic acid.

[0172] In an embodiment, the encapsulated nucleic acid is selected from any one of double-stranded DNA, single-stranded DNA, complementary DNA (cDNA), and RNA. In an embodiment, the encapsulated nucleic acid is double-stranded DNA.

[0173] In embodiments, the hydrogel beads containing the encapsulated nucleic acid bind to a dye. In embodiments, the dye intercalates between DNA bases or RNA bases. In embodiments, the dye binds to the major groove of DNA. In embodiments, the dye binds to the minor groove of DNA. In embodiments, the dye 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™, DRAQ7™, CytoPhase™ Violet, Helix NP™ Blue, Helix NP™ Green, Helix NP™ NIR, YOYO™-1, TOTO™-1 iodide (Thermo Fisher Scientific), TO-PRO-3®, SYTOX™ Blue, ethidium bromide, SYBR™ Gold, SYBR™ Green, SYBR™ Safe, EvaGreen®, and crystal violet.

[0174] One of ordinary skill in the art will be familiar with other dyes and DNA detectors that are compatible with the invention of the present disclosure in some embodiments.

[0175] IV-D. Additional Components In embodiments, the hydrogel beads described herein can include one or more additional chemical moieties. In embodiments, the hydrogel beads described herein include one or more fluorescent dyes. In embodiments, the fluorescent dye is attached to the hydrogel beads via a covalent bond. In embodiments, the fluorescent dye is attached to the hydrogel beads via non-covalent interaction. In embodiments, 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)-carboxy eosin; 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; 5-(and-6)-carboxyfluorescein succinimidyl ester; 5-(4,6-dichlorotriazinyl)aminofluorescein; 2′,7′-difluorofluorescein; eosin-5-isothiocyanate; erythrosin 5-isothiocyanate; 6-(fluorescein-5-carboxamide)hexanoic acid or succinimidyl ester; 6-(fluorescein-5-(and-6)-carboxamide)hexanoic acid or succinimidyl ester; fluorescein-S-EX succinimidyl ester; fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; Oregon Green® 488 carboxylic acid or succinimidyl ester; Oregon Green® 488 isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green® 500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidyl ester or triethylammonium salt; Oregon Green® 514 carboxylic acid; Oregon Green® 514 carboxylic acid or succinimidyl ester;Rhodamine Green (trademark) carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green (trademark) carboxylic acid, trifluoroacetamide or succinimidyl ester; Rhodamine Green (trademark)-X succinimidyl ester or hydrochloride; Rhodol Green (trademark) carboxylic acid, N,O-bis-(trifluoroacetyl) or succinimidyl ester; bis-(4-carboxypiperidinyl) sulfon rhodamine or di(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-carboxy-2’,4’,5’,7’-tetrabromosulfonfluorescein succinimidyl ester or bis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine; 6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine; 5-carboxytetramethylrhodamine succinimidyl ester; 6-carboxytetramethylrhodamine succinimidyl ester; 5-(and-6)-carboxytetramethylrhodamine succinimidyl ester; 6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester; 6-carboxy-X rhodamine succinimidyl ester; 5-(and-6)-carboxy-X rhodamine succinimidyl ester; 5-carboxy-X-rhodamine triethylammonium salt; Lissamine (trademark) rhodamine B sulfonyl chloride; malachite green; isothiocyanate; NANOGOLD (registered trademark) mono(sulfosuccinimidyl ester); QSY (registered trademark) 21 carboxylic acid or succinimidyl ester; QSY (registered trademark) 7 carboxylic acid or succinimidyl ester; Rhodamine Red (trademark)-X succinimidyl ester;6-(Tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid; succinimidyl ester; tetramethylrhodamine-5-isothiocyanate; tetramethylrhodamine-6-isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl; Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt; Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; and X-rhodamine-5-(and-6) isothiocyanate; BODIPY® dyes commercially available from Invitrogen, including but not limited to: BODIPY® FL; BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630 / 650-X STP ester; BODIPY® 650 / 665-X STP ester; 6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid; 4,4-difluoro-5,7-dimethyl-4-bora 3a,4a-diaza-s-indacene-3-pentanoic acid succinimidyl ester; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionic acid; 4,4-difluoro-5,7-dimethyl-4-bora-3a,4-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionic acid; sulfosuccinimidyl ester or sodium salt; 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3 propionyl)amino)hexanoic acid; 6-((4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; N-(4,4-difluoro 5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteic acid, succinimidyl ester or triethylammonium salt; 6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora 3a,4a 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s indacene-3-propionic acid; 4,4-difluoro-5,7-diphenyl-4-bora 3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester;4,4-Difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 6-((4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoic acid or succinimidyl ester; 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; succinimidyl ester; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid; 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid succinimidyl ester; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester; 6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoic acid or succinimidyl ester; and 6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoic acid or succinimidyl ester, Alexa Fluor dyes commercially available from Invitrogen, including but not limited to the following: Alexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid; Alexa Fluor® 488 carboxylic acid;Alexa Fluor® 532 carboxylic acid; Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid; Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid; Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid; Alexa Fluor® 660 carboxylic acid; and Alexa Fluor® 680 carboxylic acid, cyanine dyes commercially available from Amersham-Pharmacia Biotech (including, but not limited to, Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHS ester; and Cy7 NHS ester).;

[0176] In embodiments, the hydrogel beads can include 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 include 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 including the endpoints) fluorescent dyes.;

[0177] In an embodiment, the hydrogel beads contain "rainbow particles". The rainbow particles contain a mixture of fluorophores. In an embodiment, the rainbow particles contain from 1 to about 20 fluorophores, from 1 to about 10 fluorophores, or from 1 to about 5 fluorophores. In an embodiment, the hydrogel beads contain 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 sub-ranges therebetween including the ends) fluorescent dyes. In an embodiment, the user selects the wavelength to excite the rainbow particles according to the fluorophore to be investigated. The rainbow particles are commercially available, for example, from BD Biosciences (catalog numbers 556298 (mid-region 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 (e.g., catalog numbers RCP20-5 (4 colors), RCP-30-5 (6 peaks), RCP-30-5A (8 peaks)).

[0178] In an embodiment, the hydrogel beads contain a scattering control additive. In an embodiment, the scattering control additive contains polymer nanoparticles. In an embodiment, the polymer nanoparticles contain polystyrene. In an embodiment, the scattering control additive contains a comonomer. In an embodiment, the scattering control additive contains a suspension of nanoparticles.

[0179] In embodiments, the hydrogel beads are chemically functionalized hydrogel particles. In embodiments, the hydrogel contains free amine groups. In embodiments, the pre-apoptotic signal binder is attached to the free amine group. In embodiments, the pre-apoptotic signal is attached to the free amine group. In embodiments, the hydrogel beads contain allylamine. In embodiments, the hydrogel beads contain biotin. In embodiments, the hydrogel beads contain streptavidin. In embodiments, the hydrogel beads contain avidin. In embodiments, the chemically functionalized hydrogel particles contain amine groups, carboxyl groups, hydroxyl groups, or combinations thereof. In embodiments, the hydrogel beads contain a plurality of bifunctional monomers for functionalizing the hydrogel beads with different chemicals and / or molecules.

[0180] In embodiments, the hydrogel beads are functionalized to mimic one or more optical properties of target cells or labeled target cells. In embodiments, the hydrogel beads contain one or more high refractive index molecules. In embodiments, the hydrogel beads contain a plurality of high refractive index molecules. In embodiments, the high refractive index molecules enable mimicking of the SSC of target cells. In embodiments, the high refractive index molecules are selected from one or more of colloidal silica, alkyl acrylate, alkyl methacrylate, or combinations thereof. In embodiments, the high refractive index molecules are alkyl acrylate, alkyl methacrylate, or both. In embodiments, the alkyl acrylate or alkyl methacrylate contains 1 to 18, 1 to 8, or 2 to 8 carbon atoms in the alkyl group. In embodiments, the alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, 2-ethylhexyl, heptyl or octyl. In embodiments, the alkyl group is branched. In embodiments, the alkyl group is linear.

[0181] In embodiments, the hydrogel beads contain cell surface markers, epitope binding regions of cell surface markers, or combinations thereof.

[0182] V. Characteristics of Hydrogel Beads In an embodiment, the hydrogel beads described herein have artificial light scattering characteristics that are substantially similar to the corresponding light scattering characteristics of target cells.

[0183] In an embodiment, the target cells are lymphocytes, monocytes or granulocytes. In an embodiment, the target cells are prokaryotic cells. In an embodiment, the target cells are eukaryotic cells. In an embodiment, the target cells are white blood cells. In an embodiment, the target cells are platelets. In an embodiment, the target cells are red blood cells. In an embodiment, the target cells are immune cells. In an embodiment, the immune cells are T cells, B cells, NK cells, lymphocytes, monocytes, granulocytes, neutrophils, eosinophils, basophils, mast cells, macrophages, or dendritic cells.

[0184] In an embodiment, the light scattering characteristics are selected from side scatter (SSC), forward scatter (FSC), angled light scattering profile, or secondary marker profiles such as fluorescence marker profile, absorption profile, fluorescence profile or emission profile.

[0185] In an embodiment, the artificial light scattering characteristics are provided by comonomers, chemical side groups, encapsulating materials, colloidal silica, or the ratio of acrylamide to bisacrylamide.

[0186] In an embodiment, the light scattering characteristics that are substantially similar to the corresponding light scattering characteristics of the target cells are SSC. In an embodiment, the hydrogel beads have an SSC within 30%, within 25%, within 20%, within 15%, within 10%, within 5%, or within 1% of the SSC of the target cells measured by a cytometry device. In an embodiment, the side scatter of the hydrogel beads is adjusted 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.

[0187] In an embodiment, the light scattering property substantially the same as the corresponding light scattering property of the target cell is the 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 an interference method, a deviation method, a Brewster Angle method, or 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. The forward scattering can also be adjusted with lateral scattering nanoparticles that include, but are not limited to, a high density colloidal suspension of silica and / or PMMA particles with sufficient optical resolution / size / density.

[0188] In an embodiment, the refractive index (RI) of the hydrogel beads provided herein is 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.

[0189] 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.

[0190] In embodiments, the refractive index (RI) of the hydrogel beads provided herein is 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.

[0191] In embodiments, the hydrogel beads have a refractive index greater than about 1.15. In embodiments, the hydrogel beads have a refractive index greater than about 1.3. In embodiments, the hydrogel beads have a refractive index greater than about 1.7.

[0192] In some embodiments, the hydrogel particles of the present disclosure have material coefficient properties (e.g., elasticity) that are more similar to those of the target cells compared to polystyrene beads of the same diameter.

[0193] In embodiments, the dimensions (e.g., diameter, width, thickness) of the hydrogel beads of the present disclosure are substantially the same as those of the target cells. In embodiments, the hydrogel beads have a diameter of 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, less than about 800 μm, or less than about 1000 μm. In some embodiments, the hydrogel particles have a diameter greater than 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 hydrogel particles have a diameter in the range of 2.5 μm to 100 μm. In some embodiments, the hydrogel particles have a diameter of about 2.5 μm to about 25 μm, about 3 μm to about 20 μm, about 3.5 μm to about 15 μm, about 4 μm to about 12 μm, about 5 μm to about 10 μm, about 6 μm to about 9 μm, about 7 μm to about 8 μm, or about 10 μm to about 20 μm. In embodiments, the hydrogel beads have a diameter of less than about 100 μm. In embodiments, the hydrogel beads have a diameter of less than about 10 μm. In embodiments, the hydrogel beads have a diameter greater than about 10 μm. In embodiments, the hydrogel beads have a diameter of about 10 μm to about 20 μm. In embodiments, the hydrogel beads have a diameter of less than about 1 μm. In embodiments, the hydrogel beads have a diameter greater than about 1 μm. In embodiments, the diameter of the hydrogel beads is measured using dynamic light scattering.

[0194] 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 hydrogel particles have a width in the range of 2.5 μm to 100 μm. In some embodiments, the hydrogel particles have a width of about 2.5 μm to about 25 μm, about 3 μm to about 20 μm, about 3.5 μm to about 15 μm, about 4 μm to about 12 μm, about 5 μm to about 10 μm, about 6 μm to about 9 μm, or about 7 μm to about 8 μm.

[0195] 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 sub-ranges therebetween). In some embodiments, the hydrogel particles have a thickness in the range of 2.5 μm to 100 μm. In some embodiments, the hydrogel particles have a thickness of about 2.5 μm to about 25 μm, about 3 μm to about 20 μm, about 3.5 μm to about 15 μm, about 4 μm to about 12 μm, about 5 μm to about 10 μm, about 6 μm to about 9 μm, or about 7 μm to about 8.

[0196] In embodiments, when the hydrogel beads are labeled with a pre-apoptosis signal, they exhibit an MFI that is at least as large as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal.

[0197] In embodiments, when the hydrogel beads are labeled with a pre-apoptosis signal, they exhibit an MFI that is substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal.

[0198] In an embodiment, the MFI of the hydrogel beads and the MFI of the target cells are within 10% to within 50%. In an embodiment, 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 sub-ranges therebetween including the ends).

[0199] In an embodiment, the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%.

[0200] In an embodiment, when the hydrogel beads are labeled with a DNA-binding dye, they exhibit an MFI that is at least as large as the mean fluorescence intensity (MFI) of target cells labeled with the same DNA-binding dye. In an embodiment, when the hydrogel beads are labeled with a DNA-binding dye, they exhibit an MFI that is substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same DNA-binding dye.

[0201] VI. Kit Containing Hydrogel Beads In an embodiment, a kit is provided that contains the hydrogel beads of Section IV described herein.

[0202] In an embodiment, a kit comprising: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer, ii) a pre-apoptotic signal binder, and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer and ii) a pre-apoptotic signal binder, but lacks the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, wherein each bead comprises: i) a polymerizable monomer, but lacks the pre-apoptotic signal binder of the first population of hydrogel beads and ii) the encapsulated nucleic acid of the first population of hydrogel beads, is provided herein.

[0203] In an embodiment, the second and third populations of hydrogels do not contain any nucleic acids. In an embodiment, the second and third populations of hydrogels do not contain any double-stranded DNA. In an embodiment, the first, second, and third populations of hydrogel beads are in a w / w ratio of about 1:1:1. In an embodiment, the first, second, and third populations of hydrogel beads are in a ratio of about 1:1:1 in terms of the number of beads.

[0204] In an embodiment, the first, second, and third populations of hydrogel beads are in a w / w ratio of 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 an embodiment,

[0205] In an embodiment, the first population, the second population, and the third population of hydrogel beads are in a ratio of 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 terms of the number of beads.

[0206] In an embodiment, each of the first population, the second population, and the third population of hydrogel beads corresponds to about 10 - 50% of the total amount of hydrogel beads in the kit or composition by weight. In an embodiment, each of the first population, the second population, and the third population of hydrogel beads corresponds to about 10 - 50% of the total amount of hydrogel beads in the kit or composition in terms of the number of beads.

[0207] VII. Composition Containing Hydrogel Beads In an embodiment, a composition containing the hydrogel beads of Section IV described herein is provided.

[0208] In an embodiment, the composition comprises: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer, ii) a pre-apoptosis signal binder, and iii) an encapsulated nucleic acid; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer and ii) a pre-apoptosis signal binder, but lacks the encapsulated nucleic acid of the first population of hydrogel beads; and c) a third population of hydrogel beads, which comprises: i) a polymerized monomer, but lacks the pre-apoptosis signal binder of the first population of hydrogel beads and ii) the encapsulated nucleic acid of the first population of hydrogel beads.

[0209] In an embodiment, the composition comprises: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer, ii) a pre-apoptotic signal binder, and iii) an encapsulated nucleic acid; a first population of hydrogel beads; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer and ii) a pre-apoptotic signal binder, but lacks iii) the encapsulated nucleic acid of the first population of hydrogel beads; a second population of hydrogel beads; and c) a third population of hydrogel beads, which comprises i) a polymerized monomer, lacks ii) the pre-apoptotic signal binder of the first population of hydrogel beads, and lacks iii) the encapsulated nucleic acid of the first population of hydrogel beads; a third population of hydrogel beads.

[0210] In an embodiment, the composition comprises: a) a first population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer, ii) a pre-apoptotic signal, and iii) an encapsulated nucleic acid; a first population of hydrogel beads; b) a second population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer and ii) a pre-apoptotic signal, but lacks iii) the encapsulated nucleic acid of the first population of hydrogel beads; a second population of hydrogel beads; and c) a third population of hydrogel beads, which comprises i) a polymerized monomer, lacks ii) the pre-apoptotic signal of the first population of hydrogel beads, and lacks iii) the encapsulated nucleic acid of the first population of hydrogel beads; a third population of hydrogel beads.

[0211] In embodiments, the second and third populations of hydrogels do not contain any nucleic acids. In embodiments, the second and third populations of hydrogels do not contain any double-stranded DNA. In embodiments, the first, second, and third populations of hydrogel beads are in a w / w ratio of about 1:1:1. In embodiments, the first, second, and third populations of hydrogel beads are in a ratio of about 1:1:1 in terms of the number of beads. In embodiments, each of the first, second, and third populations of hydrogel beads corresponds to 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 populations of hydrogel beads corresponds to about 10-50% of the total amount of hydrogel beads in the kit or composition in terms of the number of beads.

[0212] In embodiments, the first, second, and third populations of hydrogel beads are in a w / w ratio of 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,

[0213] In embodiments, the first population, second population, and third population of hydrogel beads are in a ratio of 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 terms of the number of beads.

[0214] VIII. Methods of Using Hydrogel Beads In embodiments, methods are provided herein for determining whether a target cell sample contains one or more dead cells or pre-apoptotic cells. In embodiments, the method comprises: a) providing a population of hydrogel beads as described herein, or a population of hydrogel beads from a kit or composition provided herein; b) contacting the population of hydrogel beads 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 population of hydrogel beads; d) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the target cell sample; and e) comparing the measured concentrations of the pre-apoptotic signal and / or DNA dye in the population of hydrogel beads and the target cell sample, thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells.

[0215] In an embodiment, 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, kit, or composition described herein; b) contacting the population of hydrogel beads 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 population of hydrogel beads with a cytometry device; d) calibrating the cytometry 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. A method is provided herein.

[0216] In an embodiment, 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 described herein, or a population of hydrogel beads from a kit or composition described herein, wherein at least a subpopulation of the hydrogel beads within the population of hydrogel beads 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 population of hydrogel beads; c) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the target cell sample; and d) comparing the measured concentrations of the pre-apoptotic signal and / or DNA dye in the population of hydrogel beads and the target cell sample, thereby determining whether the target cell sample contains one or more dead cells or pre-apoptotic cells. A method is provided herein.

[0217] In an embodiment, 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 as described herein, or a population of hydrogel beads from a kit or composition as described herein, wherein at least a subpopulation of the hydrogel beads within the population of hydrogel beads 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 population of hydrogel beads with a cytometry device; c) calibrating the cytometry device based on the measured concentration of the pre-apoptotic signal and / or the DNA dye of the hydrogel beads; and d) 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. A method is provided herein.

[0218] In embodiments, the hydrogel beads provided herein can be used to determine the dynamic range and / or sensitivity of detection of specific cell surface markers or combinations thereof on a population of target cells. In embodiments, the hydrogel beads can be adjusted to have substantially the same side scatter and / or forward scatter as the target cells. In embodiments, a subpopulation of hydrogel particles is derivatized with a specific copy number of a cell surface marker. In embodiments, the cell surface marker is a cell surface receptor or a domain thereof. In embodiments, the cell surface receptor or a domain thereof is its epitope binding region. In embodiments, individual subpopulations of hydrogel beads can each be derivatized to have a unique number of copies of a cell surface marker, e.g., one subpopulation contains 100 copies of a cell surface marker, a second subpopulation contains 1,000 copies of the same cell surface marker, and a third subpopulation contains 10,000 copies of the same cell surface. In embodiments, a population of hydrogel beads is fluorescently stained for each cell surface marker, and the fluorescence is detected for the hydrogel beads in each subpopulation. In this regard, subpopulations of hydrogel particles can be used to generate a standard curve of fluorescence emission of target cells having each cell marker. The cell surface marker can be any of the provided cell surface markers, or their binding regions, or cell surface markers known to those of skill in the art.

[0219] IX. Instrument Use 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 of the present disclosure is provided in Table 2 below.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Examples

[0220] The following examples are provided for the purpose of illustrating various embodiments of the present disclosure and are not meant to limit the present disclosure in any way. Modifications and other uses of the present disclosure that are encompassed within the scope of the spirit of the present disclosure as defined by the claims will be recognized by those skilled in the art.

[0221] Example 1. Generation of Hydrogel Beads The photomask for UV lithography was supplied by CADart Services Inc. and designed using AutoCad (AutoDesk, Inc.). The SU-8 photoresist (Microchem, Inc.) was photocrosslinked on a 4-inch silicon wafer using a UV light source with a collimator (OAI, Inc.) to fabricate a master for microfluidic device manufacturing. PDMS (polydimethylsiloxane, Sigma Aldrich, Inc.) was prepared and formed using published standard methods for soft lithography and microfluidic device manufacturing (see McDonald JC, et al., 2000, Electrophoresis 21:27-40).

[0222] The droplets were formed using a flow-focusing geometry where two oil channels focused the central flow of the aqueous monomer solution to separate the droplets in the water-in-oil emulsion. Fluorocarbon oil (Novec 7500 3M, Inc.) was used as the outer continuous-phase liquid for droplet formation. To stabilize the droplets prior to polymerization, a surfactant was added to the oil phase at 0.5% w / w (ammonium carboxylate salt of Krytox 157 FSH, Dupont). To produce the basic polyacrylamide gel beads, the central phase of an aqueous monomer solution containing N-acrylamide (1 - 20% w / v), a cross-linking agent to dissolve the hydrogel (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 succinimidyl 3-(2-pyridyldithio)propionate, dicumyl alcohol dimethacrylate, dicumyl 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) was used. To induce hydrogel bead polymerization after droplet formation, an accelerator (N,N,N’,N’-tetramethylethylenediamine (2% v / v)) was added to the oil phase.

[0223] Comonomers may be added to the basic gel formulation to add functionality. Allylamine provided a primary amine group for secondary labeling after gel formation. Forward scattering can be adjusted by adjusting the refractive index of the gel by adding allyl acrylate and allyl methacrylate as comonomers. The side scattering of the droplets can be adjusted by adding a colloidal suspension (about 100 nm) of silica nanoparticles and / or PMMA (poly(methyl methacrylate)) particles to the central aqueous phase prior to polymerization.

[0224] The stoichiometric multiplexing of hydrogel beads was achieved by using comonomers containing chemically orthogonal side groups (amines, carboxyls, maleimides, epoxides, alkynes, etc.) for secondary labeling.

[0225] Droplets were formed at an average rate of 5 kHz and collected in a fluorocarbon oil phase. Polymerization was completed at 50 °C for 30 minutes, and the resulting hydrogel beads were washed from the oil into an aqueous solution.

[0226] Three different populations of hydrogel beads were prepared according to the above method. The first population of hydrogel beads contained (i) a polymerizable monomer and a bifunctional monomer, (ii) phosphatidylserine or an anti-annexin V antibody or its antigen-binding fragment (pre-apoptotic signal binder), and (iii) an encapsulated nucleic acid. This first population of hydrogel beads could bind to both pre-apoptotic signals and DNA intercalating dyes, and thus could serve as a mimic of dead cells.

[0227] The second population of hydrogel beads contained (i) a polymerizable monomer and a bifunctional monomer, and (ii) a pre-apoptotic signal binder (e.g., phosphatidylserine or an anti-annexin V antibody or its antigen-binding fragment), but lacked the encapsulated nucleic acid of the first population of hydrogel beads. This second population of hydrogel beads could bind to pre-apoptotic signals but not to DNA intercalating dyes, and thus could serve as a mimic of cells undergoing apoptosis but not yet dead.

[0228] The third population of hydrogel beads contained (i) a polymerizable monomer and a bifunctional monomer, but lacked the pre-apoptotic signal binder and the encapsulated nucleic acid of the first and second populations of hydrogel beads. This third population of hydrogel beads could bind neither to pre-apoptotic signals nor to DNA intercalating dyes, and thus could serve as a mimic of live cells.

[0229] A fourth hydrogel bead population was manufactured for the live / dead assay. This fourth population of hydrogel beads contained an amine-reactive dye binder and encapsulated nucleic acid, but did not contain the pre-apoptotic signal binder of the first population of hydrogel beads. This fourth bead population served as a dual-signal control for dead cells in cases where either a signal for DNA or amine indicated a dead population.

[0230] Example 2. Hydrogel beads mimic the forward scatter profiles and side scatter profiles of various cell types This example describes any adjustment 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 light scattering properties (e.g., forward scatter and side scatter). In some embodiments, the optical properties of the hydrogel beads are adjusted to mimic a particular cell type. The hydrogel can be adjusted by the method described in U.S. Patent No. 10,753,846. Briefly, as shown in FIGS. 74-76, the hydrogel beads are adjusted in multiple dimensions to match a particular cell type. The cells are deconvolved using optical parameters such as FSC and SSC (FIG. 75), and / or a combination of secondary markers. The hydrogel beads are further functionalized with a stoichiometrically adjusted ratio of specific chemical side groups and secondary labels, enabling the beads to exactly match the target cells without being subject to biological noise associated with the fixed cell line (FIG. 76). FIG. 1 shows that the hydrogel beads described herein can be adjusted to match the forward scatter and side scatter of various cell populations.

[0231] Example 3. Hydrogel beads can be stained with both DNA intercalating dyes and amine-reactive dyes This example demonstrates the use of the hydrogel beads of the present disclosure as controls for DNA and other amine-binding dyes. A "fourth" population of the hydrogel beads of Example 1 containing (i) a polymerizing monomer and a bifunctional monomer, (ii) an amine dye binder, and (iii) an encapsulated nucleic acid binds to both DNA intercalating dyes (Figs. 2-5) and amine-reactive dyes (Figs. 6-9). Figs. 2-5 show that the fourth population of hydrogel beads binds to multiple DNA intercalating dyes. For example, the fourth hydrogel beads bind to propidium iodide ("PI", Figs. 2-3) and 7-aminoactinomycin D ("7AAD", Figs. 4-5). Figs. 6-9 show that the fourth hydrogel beads bind to the amine-reactive dyes, GHOST DYE™ (Figs. 6-7) and LIVE / DEAD™ Fixable Blue (Figs. 8-9). Figs. 10-12 show that the fourth hydrogel beads can bind simultaneously to a DNA intercalating dye (e.g., 7AAD) and an amine-reactive dye (e.g., GHOST DYE™).

[0232] The ability of the hydrogel beads to bind to both amine-reactive dyes and DNA intercalating dyes allows the beads to serve as a control for dead cells, depending on the requirements of the assay.

[0233] Example 4. Hydrogel beads are superior to heat-killed cells and polystyrene beads as viability controls Viability controls are utilized in flow cytometry to distinguish live cells, dead cells, and apoptotic cells. (i) The hydrogel beads of Example 1 (including the fourth hydrogel bead population, the second hydrogel bead population, and the third hydrogel bead population), (ii) a mixture of heat-killed lymphocytes and live lymphocytes, and (iii) the ability of amine-reactive polystyrene beads serving as a viability control were compared. Each of (i), (ii), and (iii) was stained with both the DNA intercalating dye 7AAD and the amine-reactive dye GHOST DYE™. The concentrations of the DNA intercalating dye and GHOST DYE™ in each sample were evaluated with a cytometry device.

[0234] Figures 13 - 15 show the forward scatter and side scatter of (i) the hydrogel beads of Example 1 (Figure 14, labeled "hydrogel beads"), (ii) a mixture of heat-killed lymphocytes and live lymphocytes (Figure 13), and (iii) amine-reactive polystyrene beads (Figure 15). The (i) hydrogel beads of Example 1 showed substantially the same side scatter as lymphocytes. (Compare Figure 14 with Figure 13). In contrast, the side scatter of (iii) amine-reactive polystyrene beads was different from that of lymphocytes (compare Figure 15 with Figure 13).

[0235] Figures 16 - 17 show that the DNA intercalating dye 7AAD binds to (i) the hydrogel beads of Example 1 (Figure 17) and (ii) a mixture of heat-killed lymphocytes and live lymphocytes (Figure 16). However, (iii) amine-reactive polystyrene beads do not bind to 7AAD.

[0236] Figures 18 - 20 show that the amine-reactive dye GHOST DYE™ binds to (i) the hydrogel beads of Example 1 (Figure 19), (ii) a mixture of heat-killed lymphocytes and live lymphocytes (Figure 18), and (iii) amine-reactive polystyrene beads (Figure 20).

[0237] Unlike the amine-reactive polystyrene beads that are bound only to amine-reactive dyes, the fourth population of the hydrogel beads of Example 1 was bound to both DNA intercalating dyes and amine-reactive dyes. Thus, the hydrogel beads of Example 1 are superior to polystyrene beads as viability controls, providing scientists with versatility and a variety of options.

[0238] The hydrogel beads of Example 1 are also superior to a mixture of heat-killed lymphocytes and live lymphocytes as viability controls. The production of heat-killed lymphocyte controls and live lymphocyte controls is time-consuming because it is necessary to heat a portion of the lymphocytes to kill them. Furthermore, dead lymphocytes emit higher autofluorescence and experience higher levels of non-specific binding compared to live cells. The use of hydrogel beads reduces these problems, thereby improving consistency between experiments.

[0239] Example 5. The hydrogel beads of Example 1 mimic a target cell population containing live cells, dead cells, and cells undergoing apoptosis Compositions containing the first, second, and third populations of the hydrogel beads of Example 1 enabled the identification of live cells, dead cells, and cells undergoing apoptosis. The first hydrogel bead population contained both pre-apoptotic signal binders (e.g., phosphatidylserine (“PS”)) and encapsulated nucleic acids. The second hydrogel bead population contained pre-apoptotic signal binders but no encapsulated nucleic acids. The third hydrogel bead population contained neither pre-apoptotic signal binders nor encapsulated nucleic acids.

[0240] The first, second, and third populations of hydrogel beads were stained with annexin V tagged with fluorescein isothiocyanate (FITC) dye. This annexin V binds to PS. The hydrogel beads were also stained with the DNA intercalating dye 7AAD that intercalates between DNA bases and exhibits fluorescence. Subsequently, the three populations of the hydrogel were evaluated with a flow cytometry device.

[0241] Figure 21 shows the binding of three hydrogel bead populations to annexin V and 7AAD. The third hydrogel bead population does not bind to either annexin V or 7AAD and is labeled as a "live cell mimic". The second hydrogel bead population binds to annexin V but not to 7AAD and is labeled as an "apoptotic cell mimic". The first hydrogel bead population binds to both annexin V and 7AAD and is labeled as a "dead cell mimic".

[0242] The use of the first, second, and third populations of hydrogel beads provides an improvement over the live / dead assay of Example 4 that used the second, third, and fourth populations of hydrogel beads. The hydrogel beads of Example 4 were able to distinguish between dead cells (beads containing an amine dye binder and encapsulated nucleic acid) and live cells (lacking the amine dye binder and encapsulated nucleic acid of the fourth population).

[0243] Advantageously, the first, second, and third populations of hydrogels tested in this example serve as mimics of cell populations containing live cells, dead cells, and cells undergoing apoptosis. Live cells do not contain DNA accessible to DNA intercalating dyes and also do not have exposed PS apoptotic signal binders. Thus, the third hydrogel bead population that did not bind to either annexin V or 7AAD served as a live cell mimic. Cells undergoing apoptosis can bind to annexin V because phosphatidylserine is exposed. Thus, the second hydrogel bead population that bound to annexin V but not to 7AAD served as a mimic of cells undergoing apoptosis. Dead cells have a ruptured cell membrane and thus have exposed PS and DNA. Thus, the first hydrogel bead population that bound to both annexin V and DNA served as a mimic of dead cells.

[0244] Example 6. The hydrogel beads are stable for at least 37 days Stability of the hydrogel beads of the present disclosure. Compositions were prepared containing equal amounts of three different populations of hydrogel beads.

[0245] The first population of hydrogel beads contained (i) a polymerizable monomer and a bifunctional monomer, (ii) an anti-annexin V antibody or an antigen-binding fragment thereof (pre-apoptotic signal binder), and (iii) an encapsulated nucleic acid. This first population of hydrogel beads was able to bind to both pre-apoptotic signals and DNA intercalating dyes and could thus serve as a mimic of dead cells.

[0246] The second population of hydrogel beads contained (i) a polymerizable monomer and a bifunctional monomer and (ii) a pre-apoptotic signal binder (e.g., an anti-annexin V antibody or an antigen-binding fragment thereof), but lacked the encapsulated nucleic acid of the first population of hydrogel beads. This second population of hydrogel beads was able to bind to pre-apoptotic signals but not to DNA intercalating dyes and could thus serve as a mimic of cells undergoing apoptosis but not yet dead.

[0247] The third population of hydrogel beads contained (i) a polymerizable monomer and a bifunctional monomer, but lacked the pre-apoptotic signal binder and the encapsulated nucleic acid of the first population of hydrogel beads. This third population of hydrogel beads was unable to bind to either pre-apoptotic signals or DNA intercalating dyes and could thus serve as a mimic of live cells.

[0248] The compositions were stored at 4° C. for 37 days. The compositions were stained with annexin V dye tagged with a FITC dye that binds to the anti-annexin V antibody and the DNA intercalating dye 7AAD before storage (“day 0”) and 1, 7, 14, and 37 days after storage. The compositions were then evaluated on a flow cytometry device.

[0249] Figures 22 to 26 show the binding of the composition to annexin V and 7AAD at day 0 (Figure 22); 1 day after storage (Figure 23); 7 days (Figure 24); 14 days (Figure 25); and 37 days (Figure 26). The third hydrogel bead population did not bind to either annexin V or 7AAD during storage at 4°C for 37 days, demonstrating stability and avoidance of non-specific binding. The second hydrogel bead population bound to annexin V but not to 7AAD during storage at 4°C for 37 days. The first hydrogel bead population bound to both annexin V and 7AAD during storage at 4°C for 37 days.

[0250] This experiment showed that the hydrogel bead population remained stable and, thus, could serve as a mimic of live cells, dead cells, and apoptotic cells for at least 37 days when the hydrogel beads were stored at 4°C.

[0251] Example 7. A hydrogel bead population stained with a single dye is superior to live cells stained with a single dye as a compensation control The ability of the following compositions, each stained with a single dye, to serve as a compensation control was evaluated: (a) a composition containing the first, second, and third hydrogel bead populations of Example 1; (b) Jurkat cells; and (c) peripheral blood mononuclear cells (PBMCs).

[0252] A composition containing three hydrogel bead populations was prepared by combining the first, second, and third hydrogel bead populations in equal ratios.

[0253] Each of the following samples: (a) a composition containing three populations of hydrogel beads, (b) PBMC, and (c) Jurkat cells, was stained with either annexin V or 7AAD labeled with ALEXA FLUOR® 647 dye. The composition was then evaluated with a CYTEK® Aurora cytometry device or a BD FACSLyric™.

[0254] Figures 27-31 show the binding of 7AAD to the following samples stained with 7AAD only, using a BD FACSLyric™ cytometry device (Figures 27-29) or a CYTEK® Aurora cytometry device (Figures 30-31): (a) a composition containing three populations of hydrogel beads (Figures 27, 30), (b) Jurkat cells (Figures 28, 31), and (c) PBMC (Figure 29).

[0255] Figures 32-36 show the binding of annexin V to the following samples stained with annexin V only, using a BD FACSLyric™ cytometry device (Figures 32-34) or a CYTEK® Aurora cytometry device (Figures 35-36): (a) a composition containing three populations of hydrogel beads (Figures 32, 35), (b) Jurkat cells (Figures 33, 36), and (c) PBMC (Figure 34).

[0256] When the hydrogel bead composition was stained with a single dye (either 7AAD or annexin V), the flow cytometry scatter plots showed distinct positive (population bound to 7AAD or annexin V) and negative (population not bound to either 7AAD or annexin V) populations. In contrast, the scatter plots of cells stained with a single dye showed neither distinct positive nor negative populations (compare Figures 30 and 31). This trend was independent of the cytometry device. This indicates that the hydrogel bead composition stained with a single dye is a better control than cells stained with a single dye.

[0257] The population of hydrogel beads stained with a single dye is superior to live cells stained with a single dye as a gating control, a compensation control, and for spectral unmixing. The ability of the following compositions to serve as tools for gating control, compensation control, and spectral unmixing was evaluated: (a) a composition containing the first, second, and third populations of hydrogel beads of Example 1; (b) a composition containing Jurkat cells; and (c) a composition containing peripheral blood mononuclear cells (PBMCs).

[0258] A composition containing three populations of hydrogel beads was prepared by combining the first, second, and third populations of hydrogel beads in equal ratios.

[0259] Composition (a) was dispensed into a 96-well plate (100 μL of hydrogel beads suspended in annexin V buffer per well) and stained with annexin V labeled with 7AAD and / or ALEXA FLUOR® 647 dye.

[0260] Composition (b) was prepared by suspending Jurkat cells in annexin V buffer at a concentration of 10 million cells / mL. Composition (b) was dispensed into a 96-well plate (100 μL of composition (b) per well). Composition (b) was treated with CD95 (0.0625 μg / mL) at 37°C for 1 hour, followed by washing three times with annexin V buffer by centrifugation at 400 × g for 5 minutes. Then, composition (b) was stained with annexin V labeled with 7AAD and / or ALEXA FLUOR® 647 dye for 30 minutes.

[0261] Composition (c) was prepared by suspending PBMCs cryopreserved in annexin V buffer at a concentration of 5 million cells / mL. Composition (c) was dispensed 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 1 hour and then washed three times with annexin V buffer by centrifugation at 400×g for 5 minutes. Subsequently, composition (c) was stained with annexin V labeled with 7AAD and / or ALEXA FLUOR® 647 dye for 30 minutes.

[0262] After staining, each of compositions (a), (b) and (c) was washed three times with annexin V buffer by centrifugation at 400×g for 5 minutes.

[0263] The samples were resuspended in annexin V buffer. 500,000 cells from each sample were evaluated using a CYTEK® Aurora cytometry device or a BD FACSLyric™ cytometry device. Fluorescence spillover on the BD FACSLyric™ cytometry device was corrected by automatic compensation. Spectral unmixing was performed on the CYTEK® Aurora cytometry device using fluorescence spectral information of compositions (a), (b) and (c) labeled with a single dye (either 7AAD or annexin V labeled with ALEXA FLUOR® 647 dye).

[0264] Figures 37-39 show the forward scatter ("FSC") and side scatter ("SSC") of (a) a composition containing three populations of hydrogel beads (Figure 37); (b) a composition containing Jurkat cells (Figure 38); or (c) a composition containing peripheral blood mononuclear cells (PBMCs) (Figure 39) measured on a BD FACSLyric (trademark) cytometer device. Figures 40-42 show the forward scatter and side scatter of (a) a composition containing three populations of hydrogel beads (Figure 40); (b) a composition containing Jurkat cells (Figure 41); or (c) a composition containing peripheral blood mononuclear cells (PBMCs) (Figure 42) measured on a BD FACSLyric (trademark) cytometer device. Each composition was gated to exclude debris (see circles in Figures 37-42).

[0265] The gated cells and / or beads were analyzed on each cytometer device to determine which beads or cells bound to 7AAD and / or annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye.

[0266] Results on the BD FACSLyric (trademark) cytometer device Cells or beads from each of compositions (a), (b), and (c) bound to annexin V labeled with 7AAD and / or ALEXA FLUOR (registered trademark) 647 dye as detected on the BD FACSLyric (trademark) cytometer device. The composition (a) containing three populations of hydrogel beads was more compatible with the automatic compensation algorithm on the BD FACSLyric (trademark) cytometer device than composition (b) Jurkat cells or composition (c) PBMCs. Furthermore, the distinction between the positive and negative populations was clearer in composition (a) than in the two cell populations (b) and (c).

[0267] Figures 43 to 45 are histograms showing the mean fluorescence intensity of 7AAD in (a) a composition containing three hydrogel bead populations (Figure 43); (b) a composition containing Jurkat cells (Figure 44); or (c) a composition containing peripheral blood mononuclear cells (PBMC) (Figure 45), measured with a BD FACSLyric (trademark) cytometry device.

[0268] Figures 52 to 54 are histograms showing the mean fluorescence intensity of annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye in (a) a composition containing three hydrogel bead populations (Figure 52); (b) a composition containing Jurkat cells (Figure 53); or (c) a composition containing peripheral blood mononuclear cells (PBMC) (Figure 54), measured with a BD FACSLyric (trademark) cytometry device.

[0269] Figures 61 to 63 show the binding of annexin V labeled with 7AAD and ALEXA FLUOR (registered trademark) 647 dye to (a) a composition containing three hydrogel bead populations (Figure 61); (b) a composition containing Jurkat cells (Figure 62); or (c) a composition containing peripheral blood mononuclear cells (PBMC) (Figure 63), measured with a BD FACSLyric (trademark) cytometry device. Live cells, dead cells, and apoptotic cells (labeled "pre-apoptosis") are labeled in each figure. Fluorescence spillover on the BD FACSLyric (trademark) cytometry device was corrected by automatic compensation.

[0270] Results with the CYTEK (registered trademark) Aurora cytometry device Cells or beads from each of compositions (a), (b), and (c) were bound to annexin V labeled with 7AAD and / or ALEXA FLUOR (registered trademark) 647 dye as detected with the CYTEK (registered trademark) Aurora cytometry device.

[0271] Figures 46 to 51 are histograms showing the mean fluorescence intensity of 7AAD in (a) a composition containing three hydrogel bead populations (Figures 46, 49); (b) a composition containing Jurkat cells (Figures 47, 50); or (c) a composition containing peripheral blood mononuclear cells (PBMCs) (Figures 48, 51) measured with a CYTEK (registered trademark) Aurora cytometer device. In Figures 46 to 48, spectral unmixing was performed using the fluorescence spectrum of Jurkat cells bound to annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye. In Figures 49 to 51, spectral unmixing was performed using the fluorescence spectrum of PBMCs bound to annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye.

[0272] Figures 55 to 60 are histograms showing the mean fluorescence intensity of annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye in (a) a composition containing three hydrogel bead populations (Figures 55, 58); (b) a composition containing Jurkat cells (Figures 56, 59); or (c) a composition containing peripheral blood mononuclear cells (PBMCs) (Figures 57, 60) measured with a CYTEK (registered trademark) Aurora cytometer device. In Figures 55 to 57, spectral unmixing was performed using the fluorescence spectrum of Jurkat cells bound to annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye. In Figures 58 to 60, spectral unmixing was performed using the fluorescence spectrum of PBMCs bound to annexin V labeled with ALEXA FLUOR (registered trademark) 647 dye.

[0273] Figures 64-72 show the binding of annexin V labeled with 7AAD and ALEXA FLUOR® 647 dyes to (a) compositions containing three hydrogel bead populations (Figures 64, 67, 70); (b) compositions containing Jurkat cells (Figures 65, 68, 71); or (c) compositions containing peripheral blood mononuclear cells (PBMCs) (Figures 66, 69, 72), measured with a CYTEK® Aurora cytometer. Live cells, dead cells, and apoptotic cells (labeled "pre-apoptosis") are labeled in each figure. Spectral unmixing was performed using the fluorescence spectra of compositions containing three hydrogel bead populations (Figures 70-72), compositions containing Jurkat cells (Figures 64-66), or compositions containing PBMCs (Figures 67-69) that bound annexin V labeled with ALEXA FLUOR® 647 dye and / or 7AAD.

[0274] Conclusion This data indicates that the hydrogel bead composition can be used as a gating control, as a compensation control, and as a tool for spectral unmixing.

[0275] The hydrogel bead composition stained with pre-apoptosis signals and viability dyes (e.g., annexin V labeled with ALEXA FLUOR® 647 dye or 7AAD) shows distinct positive and negative bead populations, indicating that the hydrogel bead composition is superior to cell controls. In contrast, the separation between the positive and negative cell populations is less distinct (compare Figures 67 and 69).

[0276] The hydrogel bead composition can also regulate the number of beads that serve as live cell control mimics (i.e., beads from a third hydrogel bead population), dead cell control mimics (i.e., beads from the first hydrogel bead population), and apoptotic cell control mimics (i.e., beads from the second hydrogel bead population), and is thus superior to cell controls. In contrast, the amounts of dead cells, live cells, and apoptotic cells in a cell population that serve as a control for apoptosis cannot be precisely controlled. Thus, a hydrogel bead composition can be generated that has 33% beads each serving as dead cell mimics, live cell mimics, and apoptotic cell mimics, respectively.

[0277] The hydrogel bead composition also exhibits less variability than cells. Different lots of cells can exhibit different characteristics depending on the age of the cells. For example, the forward and side scatter of Jurkat cells of different ages are different. Compare FIG. 38 showing the forward and side scatter of 1-day-old Jurkat cells with FIG. 73 showing the forward and side scatter of 5-day-old Jurkat cells. In contrast, three replicates of the hydrogel bead population show consistent forward scatter and mean fluorescence intensity for binding to annexin V labeled with 7AAD and ALEXA FLUOR® 647 dye. These replicates are overlaid in FIGS. 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, and 67.

[0278] Furthermore, using the hydrogel bead composition as a control takes less time 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. Rather, the hydrogel bead composition is stable at 4° C. for 37 days and can be immediately stained with viability dyes.

[0279] The numbered embodiments of the present disclosure Notwithstanding the appended claims, the present disclosure describes the following numbered embodiments. 1. A hydrogel bead, comprising: a) a polymerizable monomer and a bifunctional monomer; and b) a pre-apoptotic signal binder. 2. A hydrogel bead, comprising: a) a polymerizable monomer and a bifunctional monomer; and b) a pre-apoptotic signal. 3. The hydrogel bead according to embodiment 1 or 2, wherein the pre-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 annexin V (UniProt accession number P08758), the polypeptide of SEQ ID NO: 103, the apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (UniProt accession number D9IWP9) (e.g., domain V), prothrombin or a fragment thereof (UniProt accession number P00734), milk fat globule-EGF-factor 8 (MFG-E8) (UniProt accession number Q08431), a phosphatidylserine receptor or a fragment thereof, SEQ ID NO: 102, CD36 (UniProt accession number P16671), LDL receptor-related protein (UniProt accession number P01130), or an anti-calreticulin antibody or an antigen-binding fragment thereof. 4. The hydrogel bead according to embodiment 3, wherein the pre-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof. 5. The phosphatidylserine receptor or a fragment thereof has 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 number O41514), Axl (UniProt accession number P30530), Tyro3 (UniProt accession number Q06418), Mer (UniProt accession number Q12866 or UniProt accession number Q50744), TIM-1 (also known as "KIM-1") (UniProt accession number Q96D42), TIM-4 (UniProt accession number Q96H15), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (UniProt accession number P78380), stabilin-1 (UniProt accession number Q9NY15), stabilin-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), and the hydrogel beads according to embodiment 4 comprising a polypeptide having such identity. 6. The hydrogel beads according to embodiment 1, wherein the pre-apoptosis 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 to any one of phosphatidylserine, an anti-annexin V antibody or an antigen-binding fragment thereof, annexin I (UniProt accession number P04083), calreticulin (UniProt accession number Q96L12), an anti-CD36 antibody or an antigen-binding fragment thereof, thrombospondin-1 (TSP-1) (UniProt accession number P07996), an 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, an anti-phosphatidylserine receptor or an antigen-binding fragment thereof, and an anti-LDL receptor-related protein or an antigen-binding fragment thereof. 7.c) Containing the encapsulated nucleic acid, The hydrogel beads according to any one of embodiments 1 to 6. 8. The hydrogel beads according to embodiment 7, wherein the nucleic acid is double-stranded DNA. 9. The hydrogel beads according to any one of embodiments 2 to 3, 7, and 8, wherein the pre-apoptosis signal is annexin V. 10. The hydrogel beads according to any one of embodiments 1, 3, 7, and 8, wherein the pre-apoptosis signal binder is phosphatidylserine. 11. The hydrogel beads according to any one of embodiments 1, 3, 7, and 8, wherein the pre-apoptosis signal binder is an anti-annexin V antibody or an antigen-binding fragment thereof. 12. The hydrogel beads according to any one of embodiments 2 to 3, 7, and 8, wherein the pre-apoptosis signal is apo-15. 13. The hydrogel beads according to any one of embodiments 1 to 12, wherein the hydrogel beads have artificial light scattering properties that are substantially the same as the corresponding light scattering properties of the target cell light scattering properties, and the artificial light scattering properties are provided by a comonomer, a chemical side group, an encapsulating material, colloidal silica, or a ratio of acrylamide to bisacrylamide. 14. The hydrogel beads according to any one of Embodiments 1 to 13, wherein the hydrogel beads contain a scattering control additive. 15. The hydrogel beads according to Embodiment 13, wherein the light scattering property that is substantially the same as the corresponding light scattering property of the target cells is side scatter (SSC). 16. The hydrogel beads according to Embodiment 13, wherein the light scattering property that is substantially the same as the corresponding light scattering property of the target cells is forward scatter (FSC). 17. The hydrogel beads according to Embodiment 14, wherein the scattering control additive contains a comonomer. 18. The hydrogel beads according to Embodiment 14, wherein the scattering control additive contains a suspension of nanoparticles. 19. The hydrogel beads according to any one of Embodiments 13 and 15 to 18, wherein the target cell is one of lymphocytes, monocytes, or granulocytes. 20. The hydrogel beads according to any one of Embodiments 13 and 15 to 18, wherein the target cell is one of prokaryotic cells or eukaryotic cells. 21. The hydrogel beads according to any one of Embodiments 13 and 15 to 18, wherein the target cell is a leukocyte. 22. The hydrogel beads according to any one of Embodiments 13 and 15 to 18, wherein the target cell is an immune cell. 23. The hydrogel beads according to any one of Embodiments 1 to 22, wherein the polymerizable monomer is a biodegradable monomer. 24. The hydrogel beads according to any one of Embodiments 1 to 22, wherein the hydrogel beads are biodegradable. 25. The hydrogel beads according to Embodiment 23, wherein the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. 26. The hydrogel beads according to Embodiment 23, wherein the hydrogel beads contain a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. 27. The hydrogel beads according to Embodiment 23, wherein the biodegradable monomer is a structural polysaccharide. 28. The hydrogel beads according to embodiment 23, wherein the hydrogel beads contain a structural polysaccharide. 29. The biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, alguronate, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, celluloin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, and combinations thereof; the hydrogel beads according to embodiment 23. 30. The hydrogel beads according to embodiment 23, comprising agar, agarose, alginic acid, algronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, celluloin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isomalodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof. 31. The annexin V is from human, rabbit, mouse, Ailuropoda melanoleuca, Aotus nancymaae, Balaenoptera acutorostrata scammoni, Balaenoptera musculus, Bos indicus × Bos taurus, Bos indicus, Bos mutus, Bos taurus, Bubalus bubalis, Callithrix jacchus, Camelus bactrianus, Canis lupus familiaris, Capra hircus, Carlito syrichta, Castor canadensis, cattle, Cebus imitator, Cervus canadensis, Cervus elaphus, Cervus hanglu yarkandensis, Delphinapterus leucas, Dipodomys ordii, Dipodomys spectabilis, Elephas maximus indicus, Equus przewalskii, Eschrichtius robustus, Felis catus, Gorilla gorilla, Gorilla beringer, Gulo gulo luscus, Halichoerus grypus, Hyaena hyaena, Hylobates moloch, Ictidomys tridecemlineatus, Jaculus jaculus, Lagenorhynchus obliquidens, Lemur catta, Lipotes vexillifer, Loxodonta africana, Macaca fascicularis, Macaca mulatta, Mandrillus leucophaeus, Marmota flaviventris, Marmota marmota marmota, Marmota monax, Moschus berezovskii, Muntiacus muntjak, Mustela putorius furo, Neogale vison, Neomonachus schauinslandi, Nomascus leucogenys, NyctereutesThe hydrogel beads according to any one of Embodiments 9 and 13 to 30, which are derived from a species selected from procyonoides, Odobenus rosmarus divergen, Odocoileus virginianus texanus, Orcinus orca, Ovis aries, Pan troglodytes, Papio anubis, Perognathus longimembris pacificus, Phoca vitulina, Physeter catodon, Piliocolobus tephrosceles, Propithecus coquereli, Rangifer tarandus platyrhyncus, Rhinopithecus bieti, Saimiri boliviensis boliviensis, Sciurus carolinensis, Sorex araneus, Sus scrofa, Trachypithecus francoisi, Tupaia chinensis, Tursiops truncatus, Urocitellus parryii, Ursus maritimus, Vulpes lagopus, and Zalophus californianus. 32. The hydrogel beads according to any one of Embodiments 13 and 13 to 31, wherein the annexin V comprises a sequence having at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 to 101. 33. The hydrogel beads according to any one of Embodiments 7 to 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 the group consisting of 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4′,6-diamidino-2-phenylindole (DAPI), DRAQ5™, DRAQ7™, CytoPhase™ Violet, Helix NP™ Blue, Helix NP™ Green, Helix NP™ NIR, YOYO™-1, TOTO™-1 iodide (Thermo Fisher Scientific), TO-PRO-3®, SYTOX™ Blue, ethidium bromide, SYBR™ Gold, SYBR™ Green, SYBR™ Safe, EvaGreen®, and crystal violet. 35. The hydrogel beads according to embodiment 34, wherein the dye is 7AAD. 36. The hydrogel beads according to embodiment 34, wherein the dye is propidium iodide. 37. The hydrogel beads according to any one of embodiments 1 to 36, wherein the hydrogel beads have a refractive index greater than about 1.15. 38. The hydrogel beads according to any one of embodiments 1 to 36, wherein the hydrogel beads have a refractive index greater than about 1.3. 39. The hydrogel beads according to any one of embodiments 1 to 36, wherein the hydrogel beads have a refractive index greater than about 1.7. 40. The hydrogel beads according to any one of embodiments 1 to 39, wherein the hydrogel beads have a diameter of less than about 100 μm. 41. The hydrogel beads according to any one of embodiments 1 to 39, wherein the hydrogel beads have a diameter greater than about 10 μm. 42. The hydrogel beads according to any one of embodiments 1 to 39, wherein the hydrogel beads have a diameter greater than about 1 μm. 43. The hydrogel beads according to any one of embodiments 14 to 42, wherein the scattering control additive contains polymer nanoparticles. 44. The hydrogel beads according to embodiment 43, wherein the polymer nanoparticles contain polystyrene. 45. The hydrogel beads according to any one of embodiments 1 to 44, wherein the hydrogel beads are chemically functionalized hydrogel particles. 46. The hydrogel beads according to any one of embodiments 1 to 45, wherein the hydrogel beads contain free amine groups. 47. The hydrogel beads according to embodiment 46, wherein the pre-apoptosis signal binder is attached to the free amine group. 48. The hydrogel beads according to any one of embodiments 1 to 47, wherein the hydrogel beads contain allylamine. 49. The hydrogel beads according to any one of embodiments 2 to 5, 7 to 9, and 12 to 48, which exhibit an MFI of at least the same magnitude as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal when the hydrogel beads are labeled with the pre-apoptosis signal. 50. The hydrogel beads according to any one of embodiments 2 to 5, 7 to 9, and 12 to 48, which exhibit an MFI substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal when the hydrogel beads are labeled with the pre-apoptosis signal. 51. The hydrogel beads according to any one of embodiments 1, 6 to 8, 10 to 11, and 13 - 48, which exhibit an MFI of at least the same magnitude as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal binder when the hydrogel beads are labeled with the pre-apoptosis signal binder. 52. The hydrogel beads according to any one of embodiments 1, 6 to 8, 10 to 11, and 13 - 48, which exhibit an MFI substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal binder when the hydrogel beads are labeled with the pre-apoptosis signal binder. 53. The hydrogel beads according to embodiment 50 or 52, wherein the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%. 54. The hydrogel beads according to any one of embodiments 7 to 53, wherein when the hydrogel beads are labeled with a DNA-binding dye, they exhibit an MFI at least as large as the mean fluorescence intensity (MFI) of the target cells labeled with the same DNA-binding dye. 55. The hydrogel beads according to any one of embodiments 7 to 53, wherein when the hydrogel beads are labeled with a DNA-binding dye, they exhibit an MFI substantially the same as the mean fluorescence intensity (MFI) of the target cells labeled with the same DNA-binding dye. 56. The hydrogel beads according to embodiment 55, wherein the MFI of the hydrogel beads and the MFI of the target cells are within 50%, 40%, 30%, 20% or 10%. 57. A kit comprising: a) A first population of hydrogel beads, each bead comprising: i) a polymerizable monomer; ii) a pre-apoptosis signal binder; iii) an encapsulated nucleic acid; A first population of hydrogel beads; b) A second population of hydrogel beads, each bead comprising: i) a polymerizable monomer; ii) a pre-apoptosis signal binder, but iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; A second population of hydrogel beads; and c) A third population of hydrogel beads, i) comprising a polymerizable monomer, but ii) lacking the pre-apoptosis signal binder of the first population of hydrogel beads, and iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads; A third population of hydrogel beads. A kit. 58. A composition comprising: a) A first population of hydrogel beads, wherein each bead comprises: i) A polymerized monomer; ii) A pre-apoptosis signal binder; iii) An encapsulated nucleic acid; A first population of hydrogel beads; b) A second population of hydrogel beads, wherein each bead comprises: i) A polymerized monomer; ii) A pre-apoptosis signal binder, but iii) Lacks the encapsulated nucleic acid of the first population of hydrogel beads; A second population of hydrogel beads; and c) A third population of hydrogel beads, comprising: i) A polymerized monomer, but ii) Lacks the pre-apoptosis signal binder of the first population of hydrogel beads; iii) Lacks the encapsulated nucleic acid of the first population of hydrogel beads; A third population of hydrogel beads. 59. A kit comprising: a) A first population of hydrogel beads, wherein each bead comprises: i) A polymerized monomer; ii) A pre-apoptosis signal; iii) An encapsulated nucleic acid; A first population of hydrogel beads; b) A second population of hydrogel beads, wherein each bead comprises: i) A polymerized monomer; ii) A pre-apoptosis signal, but iii) Lacks the encapsulated nucleic acid of the first population of hydrogel beads; A second population of hydrogel beads; and c) A third population of hydrogel beads, comprising: i) A polymerized monomer, but ii) lacking the pre-apoptotic signal of the first population of hydrogel beads and iii) lacking the encapsulated nucleic acid of the first population of hydrogel beads, A kit comprising a third population of hydrogel beads. 60. A composition comprising: a) A first population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer, ii) a pre-apoptotic signal, iii) an encapsulated nucleic acid, A first population of hydrogel beads; b) A second population of hydrogel beads, wherein each bead comprises: i) a polymerized monomer, ii) a pre-apoptotic signal, but iii) lacks the encapsulated nucleic acid of the first population of hydrogel beads, A second population of hydrogel beads, and c) A third population of hydrogel beads, wherein i) comprises a polymerized monomer, but ii) lacks the pre-apoptotic signal of the first population of hydrogel beads, iii) lacks the encapsulated nucleic acid of the first population of hydrogel beads, A third population of hydrogel beads, the composition comprising. 61. The kit or composition according to any one of embodiments 57 to 60, wherein the second and third populations of hydrogels do not contain any nucleic acids. 62. The kit or composition according to any one of embodiments 57 to 60, wherein the second and third populations of hydrogels do not contain any double-stranded DNA. 63. The kit or composition according to any one of embodiments 57 to 62, wherein the first, second and third populations of hydrogel beads are in a w / w ratio of about 1:1:1. 64. The kit or composition according to any one of embodiments 57 to 62, wherein the first population, the second population, and the third population of the hydrogel beads are in a ratio of about 1:1:1 in terms of the number of beads. 65. The kit or composition according to any one of embodiments 57 to 62, wherein each of the first population, the second population, and the third population of the hydrogel beads corresponds to about 10 to 50% by weight of the total amount of the hydrogel beads in the kit or the composition. 66. The kit or composition according to any one of embodiments 57 to 62, wherein each of the first population, the second population, and the third population of the hydrogel beads corresponds to about 10 to 50% in terms of the number of beads of the total amount of the hydrogel beads in the kit or the composition. 67. The kit or composition according to any one of embodiments 59 to 66, wherein the pre-apoptosis 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 annexin V (UniProt accession number P08758), the polypeptide of SEQ ID NO: 103, the apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (UniProt accession number D9IWP9) (e.g., domain V), prothrombin or a fragment thereof (UniProt accession number P00734), milk fat globule-EGF-factor 8 (MFG-E8) (UniProt accession number Q08431), a phosphatidylserine receptor or a fragment thereof, SEQ ID NO: 102, CD36 (UniProt accession number P16671), LDL receptor-related protein (UniProt accession number P01130), or an anti-calreticulin antibody or an antigen-binding fragment thereof. 68. The kit or composition according to any one of embodiments 59 to 66, wherein the pre-apoptosis signal comprises a phosphatidylserine receptor or a fragment thereof. 69. The phosphatidylserine receptor or a fragment thereof has 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 number O41514), Axl (UniProt accession number P30530), Tyro3 (UniProt accession number Q06418), Mer (UniProt accession number Q12866 or UniProt accession number Q50744), TIM-1 (also known as "KIM-1") (UniProt accession number Q96D42), TIM-4 (UniProt accession number Q96H15), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) (UniProt accession number P78380), stabilin-1 (UniProt accession number Q9NY15), stabilin-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). The kit or composition according to embodiment 68, comprising a polypeptide having such identity. 70. The pre-apoptosis signal binder according to any one of embodiments 57-58 and 61-66, and the kit or composition according to any one of embodiments 57-58 and 61-66, comprising 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 phosphatidylserine, an anti-annexin V antibody or an antigen-binding fragment thereof, annexin I (UniProt accession number P04083), calreticulin (UniProt accession number Q96L12), an anti-CD36 antibody or an antigen-binding fragment thereof, thrombospondin-1 (TSP-1) (UniProt accession number P07996), an anti-β2-glycoprotein I antibody or an antigen-binding fragment thereof, an anti-milk fat globule-EGF factor 8 (MFG-E8) or an antigen-binding fragment thereof, an anti-phosphatidylserine receptor or an antigen-binding fragment thereof, and an anti-LDL receptor-related protein or an antigen-binding fragment thereof. 71. c) comprising the encapsulated nucleic acid The kit or composition according to any one of embodiments 57-70. 72. The kit or composition according to embodiment 71, wherein the nucleic acid is double-stranded DNA. 73. The kit or composition according to any one of embodiments 59-69 and 71-72, wherein the pre-apoptosis signal is annexin V. 74. The kit or composition according to any one of embodiments 57, 58, 61-66, and 70-72, wherein the pre-apoptosis signal binder is phosphatidylserine. 75. The kit or composition according to any one of embodiments 57, 58, 61-66, and 70-72, wherein the pre-apoptosis signal binder is an anti-annexin V antibody or an antigen-binding fragment thereof. 76. The kit or composition according to any one of embodiments 59-69 and 71-72, wherein the pre-apoptosis signal is apo-15. 77. The kit or composition according to any one of embodiments 57 to 76, wherein the hydrogel beads have artificial light scattering properties that are substantially the same as the corresponding light scattering properties of the target cell light scattering properties, and the artificial light scattering properties are provided by a comonomer, a chemical side group, an encapsulating material, colloidal silica, or a ratio of acrylamide to bisacrylamide. 78. The kit or composition according to any one of embodiments 57 to 77, wherein the hydrogel beads contain a scattering adjustment additive. 79. The kit or composition according to embodiment 77 or 78, wherein the light scattering properties that are substantially the same as the corresponding light scattering properties of the target cell are side scatter (SSC). 80. The kit or composition according to embodiment 77 or 78, wherein the light scattering properties that are substantially the same as the corresponding light scattering properties of the target cell are forward scatter (FSC). 81. The kit or composition according to any one of embodiments 78 to 80, wherein the scattering adjustment additive contains a comonomer. 82. The kit or composition according to any one of embodiments 78 to 80, wherein the scattering adjustment additive contains a suspension of nanoparticles. 83. The kit or composition according to any one of embodiments 77 to 82, wherein the target cell is one of a lymphocyte, a monocyte, or a granulocyte. 84. The kit or composition according to any one of embodiments 77 to 82, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell. 85. The kit or composition according to any one of embodiments 77 to 82, wherein the target cell is a leukocyte. 86. The kit or composition according to any one of embodiments 77 to 82, wherein the target cell is an immune cell. 87. The kit or composition according to any one of embodiments 77 to 86, wherein the polymerizable monomer is a biodegradable monomer. 88. The kit or composition according to any one of embodiments 7 to 86, wherein the hydrogel beads are biodegradable. 89. The kit or composition according to embodiment 87, wherein the biodegradable monomer is a monosaccharide, disaccharide, polysaccharide, peptide, protein or protein domain. 90. The kit or composition according to embodiment 88, wherein the hydrogel beads contain a monosaccharide, disaccharide, polysaccharide, peptide, protein, or protein domain. 91. The kit or composition according to embodiment 87, wherein the biodegradable monomer is a structural polysaccharide. 92. The kit or composition according to embodiment 88, wherein the hydrogel beads contain a structural polysaccharide. 93. The biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, cellulins, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, and combinations thereof; the kit or composition according to embodiment 87. 94. The hydrogel beads are agar, agarose, alginic acid, algronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, celluloin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinitrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof, in the kit or composition of embodiment 88. 95. The annexin V is from human, rabbit, mouse, Ailuropoda melanoleuca, Aotus nancymaae, Balaenoptera acutorostrata scammoni, Balaenoptera musculus, Bos indicus × Bos taurus, Bos indicus, Bos mutus, Bos taurus, Bubalus bubalis, Callithrix jacchus, Camelus bactrianus, Canis lupus familiaris, Capra hircus, Carlito syrichta, Castor canadensis, cattle, Cebus imitator, Cervus canadensis, Cervus elaphus, Cervus hanglu yarkandensis, Delphinapterus leucas, Dipodomys ordii, Dipodomys spectabilis, Elephas maximus indicus, Equus przewalskii, Eschrichtius robustus, Felis catus, Gorilla gorilla, Gorilla beringer, Gulo gulo luscus, Halichoerus grypus, Hyaena hyaena, Hylobates moloch, Ictidomys tridecemlineatus, Jaculus jaculus, Lagenorhynchus obliquidens, Lemur catta, Lipotes vexillifer, Loxodonta africana, Macaca fascicularis, Macaca mulatta, Mandrillus leucophaeus, Marmota flaviventris, Marmota marmota marmota, Marmota monax, Moschus berezovskii, Muntiacus muntjak, Mustela putorius furo, Neogale vison, Neomonachus schauinslandi, Nomascus leucogenys, NyctereutesThe kit or composition according to any one of Embodiments 73 and 77 to 94, derived from a species selected from procyonoides, Odobenus rosmarus divergen, Odocoileus virginianus texanus, Orcinus orca, Ovis aries, Pan troglodytes, Papio anubis, Perognathus longimembris pacificus, Phoca vitulina, Physeter catodon, Piliocolobus tephrosceles, Propithecus coquereli, Rangifer tarandus platyrhyncus, Rhinopithecus bieti, Saimiri boliviensis boliviensis, Sciurus carolinensis, Sorex araneus, Sus scrofa, Trachypithecus francoisi, Tupaia chinensis, Tursiops truncatus, Urocitellus parryii, Ursus maritimus, Vulpes lagopus, and Zalophus californianus. 96. The kit or composition according to any one of Embodiments 73 and 77 to 95, wherein the annexin V comprises a sequence having at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 1 to 101. 97. The kit or composition according to any one of Embodiments 71 to 96, wherein the encapsulated nucleic acid is bound to a dye. 98. The kit or composition according to embodiment 97, 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™, DRAQ7™, CytoPhase™ Violet, Helix NP™ Blue, Helix NP™ Green, Helix NP™ NIR, YOYO™-1, TOTO™-1 iodide (Thermo Fisher Scientific), TO-PRO-3®, SYTOX™ Blue, ethidium bromide, SYBR™ Gold, SYBR™ Green, SYBR™ Safe, EvaGreen®, and crystal violet. 99. The kit or composition according to embodiment 97, wherein the dye is 7AAD. 100. The kit or composition according to embodiment 97, wherein the dye is propidium iodide. 101. The kit or composition according to any one of embodiments 77 to 100, wherein the hydrogel beads have a refractive index greater than about 1.15. 102. The kit or composition according to any one of embodiments 77 to 100, wherein the hydrogel beads have a refractive index greater than about 1.3. 103. The kit or composition according to any one of embodiments 77 to 100, wherein the hydrogel beads have a refractive index greater than about 1.7. 104. The kit or composition according to any one of embodiments 77 to 103, wherein the hydrogel beads have a diameter of less than about 100 μm. 105. The kit or composition according to any one of embodiments 77 to 103, wherein the hydrogel beads have a diameter greater than about 10 μm. 106. The kit or composition according to any one of embodiments 77 to 103, wherein the hydrogel beads have a diameter greater than about 1 μm. 107. The kit or composition according to any one of embodiments 78 to 106, wherein the scattering control additive comprises polymer nanoparticles. 108. The kit or composition according to embodiment 107, wherein the polymer nanoparticles comprise polystyrene. 109. The kit or composition according to any one of embodiments 77 to 108, wherein the hydrogel beads are chemically functionalized hydrogel particles. 110. The kit or composition according to any one of embodiments 77 to 109, wherein the hydrogel beads comprise free amine groups. 111. The kit or composition according to any one of embodiments 57, 58, 61 to 66, and 70 to 72, 74 to 75, 77 to 94, and 97 to 110, wherein the pre-apoptotic signal binder is attached to the free amine group. 112. The kit or composition according to any one of embodiments 57 to 111, wherein the hydrogel beads comprise allylamine. 113. The kit or composition according to any one of embodiments 59 to 69, 71 to 73, 76 to 110, and 112, wherein when the hydrogel beads are labeled with the pre-apoptotic signal, they exhibit an MFI at least as large as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptotic signal. 114. The kit or composition according to any one of embodiments 59 to 69, 71 to 73, 76 to 110, and 112, wherein when the hydrogel beads are labeled with the pre-apoptotic signal, they exhibit an MFI substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptotic signal. 115. The kit or composition according to any one of embodiments 57, 58, 61 to 66, and 70 to 72, 74 to 75, 77 to 94, and 97 to 112, wherein when the hydrogel beads are labeled with the pre-apoptotic signal binder, they exhibit an MFI at least as large as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptotic signal binder. 116. The 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 the hydrogel beads are labeled with the pre - apoptotic signal binder, they exhibit an average fluorescence intensity (MFI) substantially the same as that of the target cells labeled with the same pre - apoptotic signal binder. 117. The kit or composition according to 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. The kit or composition according to any one of embodiments 71 - 117, wherein when the hydrogel beads are labeled with a DNA - binding dye, they exhibit an average fluorescence intensity (MFI) at least as large as that of the target cells labeled with the same DNA - binding dye. 119. The kit or composition according to any one of embodiments 71 - 117, wherein when the hydrogel beads are labeled with a DNA - binding dye, they exhibit an average fluorescence intensity (MFI) substantially the same as that of the target cells labeled with the same DNA - binding dye. 120. The kit or composition according to 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 1 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 population of hydrogel beads 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 population of hydrogel beads; d) measuring the concentration of the pre - apoptotic signal and / or the DNA dye in the target cell sample; e) Compare the measured concentrations of the pre-apoptosis signal and / or DNA dye in the population of hydrogel beads and in the target cell sample, thereby determining whether the target cell sample contains one or more dead or pre-apoptotic cells, and a method comprising: 122. A method for determining whether a target cell sample contains one or more dead or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1 to 56, or a population of hydrogel beads from a kit or composition according to any one of embodiments 57 to 120; b) contacting the population of hydrogel beads with a pre-apoptosis signal and / or DNA dye; c) measuring the concentration of the pre-apoptosis signal and / or DNA dye in the population of hydrogel beads with a cytometry device; d) calibrating the cytometry device based on the measured concentration of the pre-apoptosis signal and / or DNA dye of the hydrogel beads; e) measuring the concentration of the pre-apoptosis signal and / or DNA dye in the target cell sample to determine whether the target cell sample contains one or more dead or pre-apoptotic cells. 123. A method for determining whether a target cell sample contains one or more dead or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1 to 56, or a population of hydrogel beads from a kit or composition according to any one of embodiments 57 to 120, wherein at least a sub-population of the hydrogel beads within the population of hydrogel beads contains a pre-apoptosis signal and / or DNA dye; b) measuring the concentration of the pre-apoptosis signal and / or DNA dye in the population of hydrogel beads; c) measuring the concentration of pre-apoptotic signals and / or DNA dyes in the target cell sample; d) comparing the measured concentration of pre-apoptotic signals and / or DNA dyes in the population of hydrogel beads and in the target cell sample, thereby determining whether the target cell sample contains one or more dead or pre-apoptotic cells, a method comprising. 124. A method for determining whether a target cell sample contains one or more dead or pre-apoptotic cells, the method comprising: a) providing a population of hydrogel beads according to any one of embodiments 1 to 56, or a population of hydrogel beads from a kit or composition according to any one of embodiments 57 to 120, wherein at least a subpopulation of hydrogel beads within the population of hydrogel beads contains pre-apoptotic signals and / or DNA dyes; b) measuring the concentration of pre-apoptotic signals and / or DNA dyes in the population of hydrogel beads with a cytometry device; c) calibrating the cytometry device based on the measured concentration of pre-apoptotic signals and / or DNA dyes of the hydrogel beads; d) measuring the concentration of pre-apoptotic signals and / or DNA dyes in the target cell sample to determine whether the target cell sample contains one or more dead or pre-apoptotic cells.

[0280] Additional Embodiments Notwithstanding the appended claims, the present disclosure describes the following additional numbered embodiments. 1. Hydrogel beads, a) a polymerizing monomer and a bifunctional monomer; b) a pre-apoptotic signal binder. 2. Hydrogel beads, comprising: a) a polymerizing monomer and a bifunctional monomer; and b) a pre-apoptotic signal. 3. The hydrogel beads according to embodiment 1 or 2, wherein the pre-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 annexin V (UniProt accession number P08758), the polypeptide of SEQ ID NO: 103, the apo-15 peptide, β2-glycoprotein 1 or a fragment thereof (UniProt accession number D9IWP9) (e.g., domain V), prothrombin or a fragment thereof (UniProt accession number P00734), milk fat globule-EGF-factor 8 (MFG-E8) (UniProt accession number Q08431), a phosphatidylserine receptor or a fragment thereof, SEQ ID NO: 102, CD36 (UniProt accession number P16671), LDL receptor-related protein (UniProt accession number P01130), or an anti-calreticulin antibody or an antigen-binding fragment thereof. 3.1. The hydrogel beads according to embodiment 3, wherein the pre-apoptotic signal comprises a phosphatidylserine receptor or a fragment thereof. 3.2. The hydrogel beads according to embodiment 4, wherein the phosphatidylserine receptor or a 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 at least one of 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), and integrin αVβ3 / β5. 4. The hydrogel beads according to Embodiment 1, wherein the pre-apoptosis signal binder is selected from the group consisting of phosphatidylserine, an anti-annexin V antibody or an antigen-binding fragment thereof, annexin I, calreticulin, an anti-CD36 antibody or an antigen-binding fragment thereof, thrombospondin-1 (TSP-1), an anti-β2-glycoprotein I antibody or an antigen-binding fragment thereof, an anti-milk fat globule-EGF-factor 8 (MFG-E8) or an antigen-binding fragment thereof, an anti-phosphatidylserine receptor or an antigen-binding fragment thereof, and an anti-LDL receptor-related protein or an antigen-binding fragment thereof. 5.c) Containing the encapsulated nucleic acid, The hydrogel beads according to any one of Embodiments 1 to 4. 6. The hydrogel beads according to Embodiment 5, wherein the nucleic acid is double-stranded DNA. 7. The hydrogel beads according to any one of Embodiments 2 to 3 and 6, wherein the pre-apoptosis signal is annexin V. 8. The hydrogel beads according to any one of Embodiments 2 to 3 and 6, wherein the pre-apoptosis signal binder is phosphatidylserine. 9. The hydrogel beads according to any one of Embodiments 1 to 3 and 6, wherein the pre-apoptosis signal binder is an anti-annexin V antibody or an antigen-binding fragment thereof. 10. The hydrogel beads according to any one of Embodiments 1 to 3 and 6, wherein the pre-apoptosis signal is apo-15. 11. The hydrogel beads according to any one of Embodiments 1 to 10, wherein the hydrogel beads have artificial light scattering properties substantially similar to the corresponding light scattering properties of the target cell light scattering properties, and the artificial light scattering properties are provided by a comonomer, a chemical side group, an encapsulating material, colloidal silica, or a ratio of acrylamide to bisacrylamide. 12. The hydrogel beads according to any one of Embodiments 1 to 10, wherein the hydrogel beads contain a scattering regulating additive. 13. The hydrogel beads according to embodiment 11, wherein the light scattering property that is substantially the same as the corresponding light scattering property of the target cells is side scatter (SSC). 14. The hydrogel beads according to embodiment 11, wherein the light scattering property that is substantially the same as the corresponding light scattering property of the target cells is forward scatter (FSC). 15. The hydrogel beads according to any one of embodiments 11 or 12, wherein the scattering-modulating additive comprises a comonomer. 16. The hydrogel beads according to any one of embodiments 11 or 12, wherein the scattering-modulating additive comprises a suspension of nanoparticles. 17. The hydrogel beads according to any one of embodiments 11 and 13 to 16, wherein the target cell is one of a lymphocyte, a monocyte, or a granulocyte. 17.1 The hydrogel beads according to any one of embodiments 11 and 13 to 16, wherein the target cell is one of a prokaryotic cell or a eukaryotic cell. 17.2 The hydrogel beads according to any one of embodiments 11 and 13 to 16, wherein the target cell is a white blood cell. 17.3 The hydrogel beads according to any one of embodiments 11 and 13 to 16, wherein the target cell is an immune cell. 18. The hydrogel beads according to any one of embodiments 1 to 17, wherein the polymerizable monomer is a biodegradable monomer. 18.1 The hydrogel beads according to any one of embodiments 1 to 17, wherein the hydrogel is biodegradable. 19. The hydrogel beads according to embodiment 18, wherein the biodegradable monomer is a monosaccharide, a disaccharide, a polysaccharide, a peptide, a protein, or a protein domain. 19.1 The hydrogel beads 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 beads according to embodiment 18, wherein the biodegradable monomer is a structural polysaccharide. 20.1 The hydrogel beads according to embodiment 18, wherein the hydrogel comprises a structural polysaccharide. 21. The biodegradable monomer is selected from the group consisting of agar, agarose, alginic acid, alguronate, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, celluloin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, and combinations thereof, and is the hydrogel beads according to Embodiment 18. 21.1 The hydrogel beads according to Embodiment 18, wherein the hydrogel comprises agar, agarose, alginic acid, algronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, carrageenan, cellodextrin, celluloin, cellulose, chitin, chitosan, chrysolaminarin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficoll, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, isomalto-dextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-linkage glucan, paramylon, pectic acid, pectin, pentastarch, phytoglycogen, prolan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinesterin, sizofiran, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof. 22. The annexin V is from human, rabbit, mouse, Ailuropoda melanoleuca, Aotus nancymaae, Balaenoptera acutorostrata scammoni, Balaenoptera musculus, Bos indicus × Bos taurus, Bos indicus, Bos mutus, Bos taurus, Bubalus bubalis, Callithrix jacchus, Camelus bactrianus, Canis lupus familiaris, Capra hircus, Carlito syrichta, Castor canadensis, cow, Cebus imitator, Cervus canadensis, Cervus elaphus, Cervus hanglu yarkandensis, Delphinapterus leucas, Dipodomys ordii, Dipodomys spectabilis, Elephas maximus indicus, Equus przewalskii, Eschrichtius robustus, Felis catus, Gorilla gorilla, Gorilla beringer, Gulo gulo luscus, Halichoerus grypus, Hyaena hyaena, Hylobates moloch, Ictidomys tridecemlineatus, Jaculus jaculus, Lagenorhynchus obliquidens, Lemur catta, Lipotes vexillifer, Loxodonta africana, Macaca fascicularis, Macaca mulatta, Mandrillus leucophaeus, Marmota flaviventris, Marmota marmota marmota, Marmota monax, Moschus berezovskii, Muntiacus muntjak, Mustela putorius furo, Neogale vison, Neomonachus schauinslandi, Nomascus leucogenys, NyctereutesHydrogel beads according to any one of embodiments 7 and 11 to 22.1, derived from a species selected from procyonoides, Odobenus rosmarus divergen, Odocoileus virginianus texanus, Orcinus orca, Ovis aries, Pan troglodytes, Papio anubis, Perognathus longimembris pacificus, Phoca vitulina, Physeter catodon, Piliocolobus tephrosceles, Propithecus coquereli, Rangifer tarandus platyrhyncus, Rhinopithecus bieti, Saimiri boliviensis boliviensis, Sciurus carolinensis, Sorex araneus, Sus scrofa, Trachypithecus francoisi, Tupaia chinensis, Tursiops truncatus, Urocitellus parryii, Ursus maritimus, Vulpes lagopus, and Zalophus californianus. 23. The hydrogel beads according to any one of embodiments 7 and 11 to 22, wherein the annexin V comprises a sequence having at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 101. 23.1. The hydrogel beads according to any one of embodiments 7 and 11 to 22, wherein the annexin V comprises a sequence having at least 100%, 95%, 90%, 85%, 80% or 75% sequence identity with the sequence of SEQ ID NO: 1. 24. The hydrogel beads according to any one of embodiments 5 to 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 the group consisting of 7-aminoactinomycin D (7AAD), propidium iodide, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4′,6-diamidino-2-phenylindole (DAPI), DRAQ5™, DRAQ7™, CytoPhase™ Violet, Helix NP™ Blue, Helix NP™ Green, Helix NP™ NIR, YOYO™-1, TOTO™-1 iodide (Thermo Fisher Scientific), TO-PRO-3®, SYTOX™ Blue, ethidium bromide, SYBR™ Gold, SYBR™ Green, SYBR™ Safe, EvaGreen®, and crystal violet. 26. The hydrogel beads according to embodiment 25, wherein the dye is 7AAD. 26.1 The hydrogel beads according to embodiment 25, wherein the dye is propidium iodide. 27. The hydrogel beads according to any one of embodiments 1 to 26.1, wherein the hydrogel beads have a refractive index greater than about 1.15. 28. The hydrogel beads according to any one of embodiments 1 to 26.1, wherein the hydrogel beads have a refractive index greater than about 1.3. 29. The hydrogel beads according to any one of embodiments 1 to 26.1, wherein the hydrogel beads have a refractive index greater than about 1.7. 30. The hydrogel beads according to any one of embodiments 1 to 29, wherein the hydrogel beads have a diameter of less than about 100 μm. 31. The hydrogel beads according to any one of embodiments 1 to 29, wherein the hydrogel bead particles have a diameter greater than about 10 μm. 32. The hydrogel beads according to any one of embodiments 1 to 29, wherein the hydrogel bead particles have a diameter greater than about 1 μm. 33. The hydrogel beads according to any one of Embodiments 11 to 32, wherein the scattering control additive contains polymer nanoparticles. 34. The hydrogel beads according to Embodiment 33, wherein the polymer nanoparticles contain polystyrene. 35. The hydrogel beads according to any one of Embodiments 11 to 34, wherein the hydrogel beads are chemically functionalized hydrogel particles. 36. The hydrogel beads according to any one of Embodiments 1 to 35, wherein the hydrogel beads contain free amine groups. 36.1 The hydrogel beads according to Embodiment 36, wherein the apoptosis signal binder is attached to the free amine group. 37. The hydrogel beads according to any one of Embodiments 1 to 35, wherein the hydrogel beads contain allylamine. 38. The hydrogel beads according to any one of Embodiments 1 and 3 to 37, wherein when the hydrogel is labeled with the pre-apoptosis signal, it exhibits an MFI at least as large as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal. 39. The hydrogel beads according to any one of Embodiments 1 and 3 to 37, wherein when the hydrogel is labeled with the pre-apoptosis signal, it exhibits an MFI substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal. 40. The hydrogel beads according to 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 beads according to any one of Embodiments 5 to 40, wherein when the hydrogel is labeled with a DNA-binding dye, it exhibits an MFI at least as large as the mean fluorescence intensity (MFI) of target cells labeled with the same DNA-binding dye. 42. The hydrogel beads according to any one of Embodiments 5 to 40, wherein when the hydrogel is labeled with the pre-apoptosis signal, it exhibits an MFI substantially the same as the mean fluorescence intensity (MFI) of target cells labeled with the same pre-apoptosis signal. 43. The hydrogel beads 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, wherein each bead comprises: i) A polymerizable monomer; ii) A pre-apoptosis signal binder; iii) An encapsulated nucleic acid; The first population of hydrogel beads; b) A second population of hydrogel beads, wherein each bead comprises: i) A polymerizable monomer; ii) A pre-apoptosis signal binder, but iii) Lacks the encapsulated nucleic acid of the first population of the hydrogel beads; The second population of hydrogel beads; and c) A third population of hydrogel beads, which i) Comprises a polymerizable monomer, but ii) Lacks the pre-apoptosis signal binder of the first population of the hydrogel beads; iii) Lacks the encapsulated nucleic acid of the first population of the hydrogel beads; The third population of hydrogel beads. 45. A composition comprising: a) A first population of hydrogel beads, wherein each bead comprises: i) A polymerizable monomer; ii) A pre-apoptosis signal binder; iii) An encapsulated nucleic acid; The first population of hydrogel beads; b) A second population of hydrogel beads, wherein each bead comprises: i) A polymerizable monomer; ii) A pre-apoptosis signal binder, but iii) Lacks the encapsulated nucleic acid of the first population of the hydrogel beads; A second population of hydrogel beads, and c) A third population of hydrogel beads, wherein i) it contains a polymerized monomer, but ii) lacks the pre-apoptosis signal binder of the first population of hydrogel beads, iii) lacks the encapsulated nucleic acid of the first population of hydrogel beads, A composition comprising a third population of hydrogel beads. 46. A composition, wherein a) A first population of hydrogel beads, wherein each bead i) contains a polymerized monomer, and ii) a pre-apoptosis signal, and iii) an encapsulated nucleic acid, A first population of hydrogel beads; b) A second population of hydrogel beads, wherein each bead i) contains a polymerized monomer, and ii) contains a pre-apoptosis signal, but iii) lacks the encapsulated nucleic acid of the first population of hydrogel beads, A second population of hydrogel beads, and c) A third population of hydrogel beads, wherein i) contains a polymerized monomer, but ii) lacks the pre-apoptosis signal of the first population of hydrogel beads, iii) lacks the encapsulated nucleic acid of the first population of hydrogel beads, A composition comprising a third population of hydrogel beads. 47. The kit or composition according to any one of embodiments 44 to 46, wherein the second population and the third population of hydrogels do not contain any nucleic acids. 48. The kit or composition according to any one of embodiments 44 to 46, wherein the second population and the third population of hydrogels do not contain any double-stranded DNA. The kit or composition according to any one of embodiments 44 to 48, wherein the first population, the second population, and the third population of the hydrogel beads are in a w / w ratio of about 1:1:1. 50. The kit or composition according to any one of embodiments 44 to 48, wherein the first population, the second population, and the third population of the hydrogel beads are in a ratio of about 1:1:1 in terms of the number of beads. 51. The kit or composition according to any one of embodiments 44 to 48, wherein each of the first population, the second population, and the third population of the hydrogel beads corresponds to about 10 to 50% by weight of the total amount of the hydrogel beads in the kit or the composition. 52. The kit or composition according to any one of embodiments 44 to 48, wherein each of the first population, the second population, and the third population of the hydrogel beads corresponds to about 10 to 50% in terms of the number of beads of the total amount of the hydrogel beads in the kit or the composition. 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 to 43, or a population of hydrogel beads from a kit or composition according to any one of embodiments 44 to 52; b) contacting the population of hydrogel beads 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 population of hydrogel beads; d) measuring the concentration of the pre-apoptotic signal and / or the DNA dye in the target cell sample; e) comparing the measured concentrations of the pre-apoptotic signal and / or the DNA dye in the population of hydrogel beads and 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 to 43, or a population of hydrogel beads from a kit or composition according to any one of embodiments 44 to 52; b) contacting the population of hydrogel beads 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 population of hydrogel beads with a cytometry device; d) calibrating the cytometry device based on the measured concentration of the pre-apoptotic signal and / or DNA dye of the hydrogel beads; 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. 55. 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 to 43, or a population of hydrogel beads from a kit or composition according to any one of embodiments 44 to 52, wherein at least a sub-population of the hydrogel beads within the population of hydrogel beads 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 population of hydrogel beads; c) measuring the concentration of the pre-apoptotic signal and / or DNA dye in the target cell sample; d) comparing the measured concentrations of the pre-apoptotic signal and / or DNA dye in the population of hydrogel beads and the target cell sample; A method comprising 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 to 43, or a population of hydrogel beads from a kit or composition according to any one of embodiments 44 to 52, wherein at least a subpopulation of the hydrogel beads within the population of hydrogel beads 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 population of hydrogel beads with a cytometry device; c) calibrating the cytometry device based on the measured concentration of the pre-apoptotic signal and / or the DNA dye of the hydrogel beads; d) 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.

[0281] Incorporation by reference All references, papers, publications, patents, patent publications, and patent applications cited in this specification are hereby incorporated by reference in their entirety for all purposes. However, any reference to any reference, paper, publication, patent, patent publication, and patent application cited in this specification is not an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world, nor should they be construed as such. Further, U.S. Patent No. 9,915,598, entitled "Hydrogel Particles with Tunable Optical Properties," issued on March 13, 2018, is hereby incorporated by reference in this specification for all purposes. Further, U.S. Patent No. 9,714,897, entitled "Hydrogel Particles with Tunable Optical Properties and Methods for Using the Same," issued on July 25, 2017, is hereby incorporated by reference in this specification for all purposes. Further, U.S. Patent No. 11,313,782, entitled "Compositions and Methods for Cell-Like Calibration Particles," issued on April 26, 2022, is hereby incorporated by reference in this specification for all purposes. Further, U.S. Patent Application Publication No. 2023 / 0067460, entitled "Hydrogel Particles as Feeder Cells and Syntehtic Antigen Presenting Cells," published on March 2, 2023, is hereby incorporated by reference in this specification for all purposes. Further, International Application No. PCT / US2023 / 066684, entitled "Engineered Particles as Red Blood Cell Mimics and Compositions Containing Same for Hematology," filed on May 5, 2023, is hereby incorporated by reference in this specification for all purposes.Furthermore, International Publication No. WO 2021 / 226036, entitled Compositions and Methods for Passive Optical Barcoding for Multiplexed Assays, published on November 11, 2021, is incorporated herein by reference for all purposes.

Claims

1. a) A first group of hydrogel beads, wherein each hydrogel bead in the first group of hydrogel beads is i) Polymerization monomers and ii) Pre-apoptotic signal binders, iii) Double-stranded DNA and, The first group of hydrogel beads, including the above, b) A second group of hydrogel beads, wherein each hydrogel bead in the second group of hydrogel beads is i) Polymerization monomers and ii) Includes a pre-apoptotic signal binder, iii) The first group of hydrogel beads lacks double-stranded DNA, The second group of the above hydrogel beads, and c) A third group of hydrogel beads, wherein each hydrogel bead in the third group of hydrogel beads is i) Contains polymerization monomers, ii) The first and second populations of hydrogel beads lack pre-apoptotic signaling binders, iii) The first group of hydrogel beads lacks double-stranded DNA, The third group of hydrogel beads mentioned above, A kit that includes this.

2. The kit according to claim 1, wherein the pre-apoptotic signal binder comprises a polypeptide having at least 75% identity with one of the following: 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, or anti-LDL receptor-related protein or its antigen-binding fragment.

3. The kit according to claim 2, wherein the pre-apoptotic signal binder comprises a polypeptide having at least 75% identity with an anti-annexin V antibody or its antigen-binding fragment.

4. The kit according to claim 3, wherein the pre-apoptotic signaling binder comprises phosphatidylserine.

5. The kit according to claim 1, wherein the pre-apoptotic signal binder can bind to a pre-apoptotic signal comprising a polypeptide having at least 75% identity with one of the following: a phosphatidylserine receptor or a fragment thereof, apo-15 peptide, β2-glycoprotein 1 or a fragment thereof, prothrombin or a fragment thereof, milk fat globule-EGF factor 8 (MFG-E8), CD36, LDL receptor-related protein, anti-calreticulin antibody or an antigen-binding fragment thereof, or the polypeptide of SEQ ID NO:

103.

6. The kit according to claim 5, wherein the pre-apoptotic signal comprises a polypeptide having at least 75% identity with a phosphatidylserine receptor or a fragment thereof.

7. The kit according to claim 6, wherein the pre-apoptotic signal comprises annexin V, which contains a polypeptide having at least 75% sequence identity with SEQ ID NO:

102.

8. The kit according to claim 1, wherein the double-stranded DNA of the first population of hydrogel beads can be bound to a DNA intercalation dye.

9. The kit according to claim 1, wherein the first group of hydrogel beads further comprises an amine dye binder capable of binding to an amine dye.

10. The kit according to claim 1, wherein one hydrogel bead from a first, second, or third population of hydrogel beads has artificial light scattering properties substantially similar to those of the target cell.

11. The kit according to claim 10, wherein the artificial light scattering properties substantially similar to those of the target cells are side scattering (SSC) or forward scattering (FSC).

12. One or more polymerization monomers of the first, second, or third group of hydrogel beads are agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capslan, carrageenan polysaccharide, cellodextrin, cerulin, cellulose, chitin, chitosan, chrysolaminalin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficol, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, The kit according to claim 1, comprising glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-bound glucan, paramylon, pectinic acid, pectin, pentastarch, phytoglycogen, proylan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, schizophyllan, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

13. The kit according to claim 1, wherein one or more polymerization monomers of the first, second, or third group of hydrogel beads contain polyacrylamide.

14. a) A first group of hydrogel beads, wherein each hydrogel bead in the first group of hydrogel beads is i) Polymerization monomers and ii) Double-stranded DNA and amine dye binder, The first group of hydrogel beads, including, b) A second group of hydrogel beads, wherein each hydrogel bead in the second group of hydrogel beads is i) Contains polymerization monomers, ii) The first population of hydrogel beads lacks double-stranded DNA and amine dye binder. The second group of hydrogel beads mentioned above, A kit that includes this.

15. The kit according to claim 14, wherein the double-stranded DNA of the first population of hydrogel beads can bind to the DNA intercalation dye, and the amine dye binder can bind to the amine dye.

16. The kit according to claim 14, wherein the first and / or second population of hydrogel beads has artificial light scattering properties substantially similar to those of the target cells.

17. The kit according to claim 16, wherein the artificial light scattering properties substantially similar to those of the target cells are side scattering (SSC) or forward scattering (FSC).

18. One or more polymerization monomers of the first and / or second group of hydrogel beads are agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabinoxylan, beta-glucan, callose, capslan, carrageenan polysaccharide, cellodextrin, cerulin, cellulose, chitin, chitosan, chrysolaminalin, curdlan, cyclodextrin, alpha-cyclodextrin, dextrin, dextran, ficol, fructan, fucoidan, galactoglucomannan, galactomannan, galactosaminogalactan, gellan gum, glucan, The kit according to claim 14, comprising glucomannan, glucuronoxylan, glycocalyx, glycogen, hemicellulose, homopolysaccharide, hypromellose, icodextrin, inulin, kefiran, laminarin, lentinan, levan polysaccharide, lichenin, mannan, mixed-bonded glucan, paramylon, pectinic acid, pectin, pentastarch, phytoglycogen, proylan, polydextrose, polysaccharide peptide, porphyran, pullulan, schizophyllan, sinistrin, schizophyllan, welan gum, xanthan gum, xylan, xyloglucan, zymosan, or a combination thereof.

19. The kit according to claim 14, wherein one or more polymerization monomers of the first and / or second group of hydrogel beads contain polyacrylamide.

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