A method for simultaneously identifying a subpopulation of cells and the phosphorylation level of a related factor

Abstract

The application discloses a method for simultaneously identifying cell subgroups and related factor phosphorylation levels. The method simplifies the protein phosphorylation flow cytometry staining step without affecting the staining effect, can simultaneously detect 17 stained cells, and simultaneously saves cost and is more efficient. The method provided by the application can provide more accurate distinguishing ability, can obtain more information from a small cell sample, can detect abnormal cells at a low level, increases laboratory efficiency, is especially suitable for the research of the immune system, and has a wide prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for simultaneously identifying cell subpopulations and the phosphorylation levels of related factors. Background Technology

[0002] Protein phosphorylation is the process of transferring a phosphate group to a substrate protein under the catalysis of a kinase. Phosphorylation mainly occurs on tyrosine, serine, and threonine residues of the substrate protein, causing conformational changes in the protein. This dynamically regulates protease activity and protein-protein interactions, leading to a sequential protein phosphorylation cascade that rapidly transmits signals, ultimately regulating various events closely related to life activities, such as cell metabolism, growth, proliferation, and apoptosis. Diseases such as cancer, infection, and inflammation are often accompanied by abnormal protein phosphorylation events. Therefore, a deeper understanding of protein phosphorylation processes and signaling pathways is crucial for further research on their role in diseases.

[0003] Traditionally, the immunoblotting method is used to detect the phosphorylation level of molecules in the JAK-STAT pathway. This method is time-consuming and complex, making it unsuitable for rapid, accurate, and quantitative detection of protein expression levels. Furthermore, it cannot simultaneously detect the phosphorylation level of molecules in the JAK-STAT pathway in different cellular subpopulations.

[0004] Therefore, it is of great importance in this field to provide an analytical method that can simultaneously identify the phosphorylation levels of various cellular subpopulations and their signal transduction and transcriptional activator families. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a method for simultaneously identifying cell subpopulations and the phosphorylation levels of related factors.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a method for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, the method comprising: identifying cell subpopulations using a first antibody mixture, and then detecting phosphorylation levels of related factors using a second antibody mixture, wherein the antibodies in the first antibody mixture include one or more of CD183, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, and CD197, and the antibodies in the second antibody mixture include one or more of STAT1, STAT3, STAT4, STAT5, and STAT6.

[0008] Furthermore, the antibody-coupled fluorescent dye in the first antibody mixture includes one or more of BUV395, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, Alexa Fluor 700, and APC-Cy7.

[0009] Furthermore, the ratio of antibodies conjugated with fluorescent dyes is as follows: CD183 BUV395: CD8a BV510: CD127BV605: CD45RA BV650: CD279 BV711: CCR6 PE: CD185 PE / Dazzle 594: CD25 PECy5: CD197APC-Cy7 = 1:1:2:2:1:1:1:1:2.

[0010] Furthermore, the second antibody mixture contains an antibody-coupled fluorescent dye, the fluorescent dye including one or more of AF488, BV421, APC, PECy7, and PerCP-Cy5.5.

[0011] Furthermore, the ratio of antibodies conjugated with fluorescent dyes is: STAT1 AF488 : STAT3 BV421 : STAT4 APC : STAT5 PECy7 : STAT6 PerCP-Cy5.5 = 1:1:1:1:1.

[0012] Furthermore, the staining system used to identify cell subpopulations using the first antibody mixture included PBS, BSA, EDTA, and sodium azide.

[0013] Furthermore, the staining system contains 1×PBS, 0.2% BSA, 2mM EDTA, and 0.1% sodium azide.

[0014] Furthermore, the amount of the staining system used is per 1×10 6 50 μL per cell.

[0015] Furthermore, the identification of cell subpopulations using the first antibody mixture also includes pre-stained cells.

[0016] Furthermore, cells were fixed using BD phosflow Fix Buffer I.

[0017] Furthermore, the method also includes incubating the cells with interleukin before fixing them.

[0018] Furthermore, the interleukin is a human interleukin.

[0019] Furthermore, the human interleukins include IL-2, IL-6, and IL-12.

[0020] Furthermore, the concentration ratio of IL-2, IL-6, and IL-12 is 20:4:1.

[0021] Furthermore, the method also includes penetrating the cell biomembrane after fixing the cells.

[0022] Furthermore, the method for penetrating cell biomembranes involves centrifuging, precipitating, resuspending, and washing the cells.

[0023] Furthermore, the cells were resuspended using BD phosflow Perm Buffer III.

[0024] Further, the cells were washed using BD Pharmingen Stain Buffer.

[0025] Furthermore, the staining system used to identify cell subpopulations using the second antibody mixture included PBS, BSA, EDTA, and sodium azide.

[0026] Furthermore, the method also includes washing and resuspending the cells after staining.

[0027] Further, the cells were washed using Pharmingen Stain Buffer.

[0028] Furthermore, the cells were resuspended using PBS, BSA, EDTA, and sodium azide.

[0029] Furthermore, the concentrations of PBS, BSA, EDTA, and sodium azide are 1×PBS, 0.2% BSA, 2mM EDTA, and 0.1% sodium azide, respectively.

[0030] Furthermore, the cells are T lymphocytes.

[0031] Furthermore, the sample is a PBMC.

[0032] Furthermore, the method also includes analyzing the processed samples using a flow cytometer.

[0033] Furthermore, the flow cytometer is a multicolor flow cytometer.

[0034] A second aspect of the present invention provides a reagent for simultaneously identifying cell subsets and phosphorylation levels of related factors in a sample, the reagent comprising: a first antibody mixture and a second antibody mixture, wherein the antibodies in the first antibody mixture include one or more of CD183, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, and CD197, and the antibodies in the second antibody mixture include one or more of STAT1, STAT3, STAT4, STAT5, and STAT6.

[0035] Furthermore, the first antibody mixture and the second antibody mixture contain antibody-coupled fluorescent dyes.

[0036] Furthermore, the antibody-conjugated fluorescent dye in the first antibody mixture includes one or more of BUV395, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, Alexa Fluor 700, and APC-Cy7.

[0037] Furthermore, the ratio of antibodies conjugated with fluorescent dyes is as follows: CD183 BUV395: CD8a BV510: CD127BV605: CD45RA BV650: CD279 BV711: CCR6 PE: CD185 PE / Dazzle 594: CD25 PECy5: CD197APC-Cy7 = 1:1:2:2:1:1:1:1:2.

[0038] Furthermore, the antibody-conjugated fluorescent dye in the second antibody mixture includes one or more of AF488, BV421, APC, PECy7, and PerCP-Cy5.5.

[0039] Furthermore, the ratio of antibodies conjugated with fluorescent dyes is: STAT1 AF488 : STAT3 BV421 : STAT4 APC : STAT5 PECy7 : STAT6 PerCP-Cy5.5 = 1:1:1:1:1.

[0040] Furthermore, the reagent also includes a staining system.

[0041] Furthermore, the staining system includes PBS, BSA, EDTA, and sodium azide.

[0042] Furthermore, the staining system contains 1×PBS, 0.2% BSA, 2mM EDTA, and 0.1% sodium azide.

[0043] Furthermore, the reagent also includes interleukin.

[0044] Furthermore, the interleukin is a human interleukin.

[0045] Furthermore, the human interleukins include IL-2, IL-6, and IL-12.

[0046] Furthermore, the concentration ratio of IL-2, IL-6, and IL-12 is 20:4:1.

[0047] Furthermore, the reagents also include one or more of BD phosflow Fix Buffer I, BD phosflow Perm Buffer III, and BD Pharmingen Stain Buffer.

[0048] Furthermore, the cells are T lymphocytes.

[0049] Furthermore, the sample is a PBMC.

[0050] A third aspect of the present invention provides a kit for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, the kit comprising the reagents described in the second aspect of the present invention.

[0051] Furthermore, the cells are T lymphocytes.

[0052] Furthermore, the kit also includes packaging materials for packaging the composition.

[0053] Furthermore, the kit also includes instructions.

[0054] A fourth aspect of the present invention provides a system for simultaneously identifying cell subsets and phosphorylation levels of related factors in a sample, the system comprising:

[0055] A memory that stores processor-readable instructions; and one or more processors arranged to read and execute the instructions stored in the memory;

[0056] The processor-readable instructions include instructions arranged to control the computer to perform the method described in the first aspect of the present invention.

[0057] Furthermore, the cells are T lymphocytes.

[0058] The fifth aspect of the present invention provides the application of the method described in the first aspect of the present invention, the reagent described in the second aspect of the present invention, the kit described in the third aspect of the present invention, or the system described in the fourth aspect of the present invention in identifying cell subpopulations and phosphorylation levels of related factors in a sample.

[0059] Furthermore, the relevant factors include one or more of STAT1, STAT3, STAT4, STAT5, and STAT6.

[0060] Furthermore, the cells are T lymphocytes.

[0061] Advantages and beneficial effects of the present invention:

[0062] This application simplifies the protein phosphorylation flow cytometry staining procedure without affecting the staining effect, enabling simultaneous detection of 17 types of stained cells. Furthermore, it saves costs and is more efficient. The method provided in this application offers more precise discrimination capabilities, can obtain more information from small cell samples, can detect abnormal cells at low levels, increases laboratory efficiency, and is particularly suitable for immune system research, showing broad prospects. Attached Figure Description

[0063] Figure 1 This is a diagram illustrating the phylogenetic strategy of T lymphocytes stained with a 17-color reagent kit. 1A is a diagram of the lymphocyte population after excluding double adhesions, debris, and dead cells. 1B is a diagram distinguishing between the phylogenetic strategy and cell subsets after staining the surface of CD4+ T lymphocytes in T lymphocytes. 1C is a diagram distinguishing between the phylogenetic strategy and cell subsets after staining the surface of CD8+ T lymphocytes in T lymphocytes.

[0064] Figure 2 The diagram shows the effect of changes in the staining, fixation, and permeabilization sequence on cell surface markers and protein phosphorylation levels. In the diagram, 2A shows the sequence of staining, fixation, and permeabilization, and 2B shows the sequence of fixation, staining, and permeabilization.

[0065] Figure 3 This is a flow cytometry graph showing the expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway of CD4+ T lymphocytes (significant changes are indicated by %), with gray representing isotype controls and colored representing phosphorylation. Graph 3A shows the expression level of pSTAT1 in CD4+ T lymphocytes, 3B shows the expression level of pSTAT3 in CD4+ T lymphocytes, 3C shows the expression level of pSTAT4 in CD4+ T lymphocytes, 3D shows the expression level of pSTAT5 in CD4+ T lymphocytes, and 3E shows the expression level of pSTAT6 in CD4+ T lymphocytes.

[0066] Figure 4This is a graph showing the expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway of CD8+ T lymphocytes using flow cytometry for protein phosphorylation (significant changes are indicated by %), with gray representing isotype controls and colored representing phosphorylation. Detailed Implementation

[0067] The following provides definitions for some of the terms used in this specification. Unless otherwise stated, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] This invention provides a method for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample. The method includes: identifying cell subpopulations using a first antibody mixture, and then detecting phosphorylation levels of related factors using a second antibody mixture. The antibodies in the first antibody mixture include one or more of CD183, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, and CD197. The antibodies in the second antibody mixture include one or more of STAT1, STAT3, STAT4, STAT5, and STAT6.

[0069] In some embodiments, the method of this application can identify multiple specific cell types and simultaneously identify the phosphorylation levels of related factors. The cell types include T lymphocytes, which include CD4+ and CD8+ T cells, further subdivided into naive T cells (TN), central memory cells (TCM), effector memory T cells (TEM), and regulatory T cells (Treg). Based on the characteristics of each subset of CD4+ and CD8+ T cells, CD4+ T cells are further subdivided into naive T cells (TN), central memory cells (TCM), effector memory T cells (TEM), helper T cells 1 (Th1), helper T cells 2 (Th2), helper T cells 17 (Th17), follicular helper T cells (Tfh), and regulatory T cells (Treg); CD8+ T cells are further subdivided into naive T cells (TN), central memory cells (TCM), effector memory T cells (TEM), and regulatory T cells (Treg). The phosphorylated molecules include STAT1, STAT3, STAT4, STAT5, and STAT6.

[0070] In some embodiments, the method of this application can simultaneously identify the levels of 17 cell surface markers and 5 phosphorylation molecules. The presence or absence of the 17 cell surface markers and 5 phosphorylation molecules is determined simultaneously (i.e., at the same time). Using the method of this application, a single analysis can be performed on a cell population, thereby determining the presence or absence of the 17 markers and 5 phosphorylation molecules. It is not necessary to run more than one analysis or analyze multiple samples.

[0071] In some embodiments, antibodies capable of specifically binding to various cell surface markers are known in the art. An antibody specifically binds to a cell surface marker sequence when it binds with preferential or high affinity to that sequence but substantially does not bind, does not bind, or only binds with low affinity to other cell surface markers or other proteins.

[0072] In some embodiments, each antibody is typically labeled with a different fluorescent label. The antibodies in the first antibody mixture (CD183, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, CD197) are conjugated with the fluorescent dyes BUV395, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, AlexaFluor 700, and APC-Cy7, respectively. The antibodies in the second antibody mixture (STAT1, STAT3, STAT4, STAT5, STAT6) are conjugated with the fluorescent dyes AF488, BV421, APC, PECy7, and PerCP-Cy5.5, respectively.

[0073] In some embodiments, each of the fluorescently labeled antibodies in this application is typically titrated to an appropriate concentration for use in the methods of this application, with each antibody typically titrated at a ratio of 1:10 to 1:1,000,000, such as 1:50, 1:100, 1:200, 1:300, 1:500, 1:1,000, 1:10,000, 1:50,000, or 1:100,000.

[0074] In some embodiments, the cell population is typically present in the sample. The sample is preferably a liquid sample. The sample can be urine, lymph, saliva, mucus, milk, or amniotic fluid, but is preferably blood, plasma, or serum. In this application, the sample is PBMCs. Freshly prepared PBMCs are generally used. If frozen PBMCs are used, the conditions for cell recovery after thawing should be optimized to ensure adequate basal phosphorylation levels in unstimulated cells and appropriate cellular responses to stimulation.

[0075] Samples are typically processed before testing, for example by centrifugation or by passing them through a membrane that filters out unwanted molecules or cells (such as red blood cells). Samples can be measured immediately after collection. Alternatively, samples can usually be stored before testing, preferably at temperatures below -70°C.

[0076] This invention provides a kit for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample.

[0077] In some embodiments, the components of the kit may be packaged in an aqueous medium or in a lyophilized form. Suitable containers in the kit typically include at least one vial, test tube, long-necked flask, PET bottle, syringe, or other container in which one component can be placed, and preferably, appropriately aliquoted. When more than one component is present in the kit, the kit will also typically include a second, third, or other additional container in which the additional components are placed separately. However, different combinations of components may be contained in a single vial. The kit of the present invention will also typically include a container for containing the reactants, sealed for commercial sale. Such a container may include injection-molded or blow-molded plastic containers in which the desired vials can be held.

[0078] The solid support of the kit can be plastic, silicon wafer, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate or slide.

[0079] The kit of this application may additionally include one or more other reagents or instruments that enable any of the embodiments mentioned above. The reagents or instruments include one or more of the following: a suitable buffer solution (aqueous solution), a device for obtaining samples from the subject (e.g., a container or instrument including a needle), and / or other reagents required for FACS analysis. The reagents may be present in the kit in a dry state, allowing them to be resuspended in a liquid sample. The kit may also optionally include instructions for use in the methods of this application or detailed information about the types of samples for which the methods can be used.

[0080] This invention provides a system for simultaneously identifying cell subsets and phosphorylation levels of related factors in a sample, the system comprising:

[0081] Memory that stores processor-readable instructions; and

[0082] One or more processors are configured to read and execute instructions stored in the memory; wherein the processor-readable instructions include instructions configured to control a computer to perform the methods described above.

[0083] In this invention, the implementation of the system may include performing or completing selected tasks manually, automatically, or in combination thereof. Furthermore, the actual instruments and equipment according to embodiments of the system of this invention can implement multiple selected tasks via hardware, software, firmware, or a combination thereof using an operating system.

[0084] The invention is further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are by way of example and are not intended to limit the invention. The main features of the invention can be used in various embodiments without departing from the scope of the invention.

[0085] Example

[0086] 1. Experimental materials

[0087] The fluorescently labeled antibodies used in this application are specifically binding monoclonal antibodies. The names, manufacturers, clone numbers, and antibody titers of the fluorescent antibodies are shown in Table 1. The surface marker antibody titer is determined based on the stability of the binding between the cell surface marker and the fluorescein, and whether the fluorescently labeled antibody of the cell surface marker is resistant to the fixative and permeabilizing agents.

[0088] Table 1 Antibodies

[0089]

[0090]

[0091] PBMC: derived from rheumatoid arthritis patients collected at Beijing Hospital in May 2025.

[0092] Instruments and equipment:

[0093] BD LSRFortessa™ Flow Cytometer (BD Company, Part No.: 647800)

[0094] Centrifuge 5801 R centrifuge (Eppendorf GmbH, Germany)

[0095] Centrifuge 5417 R centrifuge (Eppendorf GmbH, Germany)

[0096] Manual defrosting refrigerator (Haier Company)

[0097] -80°C ultra-low temperature freezer (Thermo Scientific)

[0098] 37°C water bath

[0099] 30-300µL multichannel pipettes (Eppendorf)

[0100] 1-10µL pipettes (Eppendorf)

[0101] 100µL pipette (Eppendorf)

[0102] 1000µL pipette (Eppendorf)

[0103] Reagents and consumables:

[0104] 5ml round-bottom polystyrene flow cytometer (Falcon, part number: 352008)

[0105] 40µm blue sterile cell filter (Falcon, part number: 35234)

[0106] 50ml sterile centrifuge tubes (Sorfa, product number: t124-4)

[0107] 15ml centrifuge tubes (ServiceBio, part number: ep-1500)

[0108] 1.5ml centrifuge tubes (ServiceBio, part number: ep-150)

[0109] 2ml centrifuge tubes (ServiceBio, part number: ep-200)

[0110] 200µL centrifuge tubes (Axygen, part number: pcr-02-c)

[0111] Transparent polystyrene 96V perforated plate 3897 (Corning Company, part number: 07-200-108)

[0112] Lymphocyte separation medium, 500ml (Alere Technolgoies AS, catalog number: AXS-1114546)

[0113] Dimethyl sulfoxide (Sigma-Aldrich, product number: D2650-100ML)

[0114] Fetal bovine serum, 500ml (Sciencell, catalog number: 0500)

[0115] RPMI 1640 culture medium (gibco, catalog number: 11875093)

[0116] Cell stimulating factors

[0117] Phosphate-buffered saline (1×PBS), (HyClone, catalog number: SH30256.01)

[0118] BD phosflow Perm Buffer III (Item No.: 558050)

[0119] BD Phosflow Fix Buffer I (Item No.: 557870)

[0120] BD Pharmingen Stain Buffer (FBS) (Item No.: 554656)

[0121] Fluorescent protein-conjugated antibodies (BD, Biolegend, eBioscience)

[0122] Reagent preparation

[0123] (1) BD phosflow Fix Buffer I was incubated in a water bath at 37 degrees Celsius in advance, and BD phosflow PermBuffer III was incubated in a refrigerator at -20 degrees Celsius in advance.

[0124] (2) RPMI 1640 medium was prepared into a complete medium containing 10% FBS.

[0125] (3) Red blood cell lysis buffer: 1.0 g potassium bicarbonate (KHCO3), 8.3 g ammonium chloride (NH4Cl), 0.037 g EDTA-Na2, add double H2O to 1000 ml to make working solution.

[0126] 2. Experimental Methods

[0127] (1) Preparation of human peripheral blood mononuclear cells (PBMCs)

[0128] Dilute the blood (without plasma and anticoagulated) to a volume ratio of 1:1 (blood:1×PBS). Inject the diluted blood into the upper layer of the lymphocyte separation medium at a ratio of 2:1 (blood:lymphocyte separation medium). Centrifuge at 500g, 20°C for 30 minutes, adjusting the centrifuge speed to 3 units for the ascent and 2 units for the descent. Aspirate the white, flocculent mononuclear cells and resuspend them in 4 times the volume of RPMI 1640. Wash twice by centrifugation at 600g, 6 minutes. If the aspirated mononuclear cells contain a large number of red blood cells, wash thoroughly using 10mL of peripheral blood with 1mL of red blood cell lysis buffer at room temperature for 3 minutes, followed by centrifugation with 5 times the volume of 1×PBS.

[0129] (2) Flow cytometry staining of surface proteins

[0130] T lymphocyte subsets were identified based on cell surface markers. T lymphocytes include CD4+ and CD8+ T cells, further divided into naive T cells (TN), central memory cells (TCM), effector memory T cells (TEM), and regulatory T cells (Treg). Based on the characteristics of each CD4+ and CD8+ T cell subset, CD4+ T cells were further subdivided into naive T cells (TN), central memory cells (TCM), effector memory T cells (TEM), helper T cells 1 (Th1), helper T cells 2 (Th2), helper T cells 17 (Th17), follicular helper T cells (Tfh), and regulatory T cells (Treg); CD8+ T cells were subdivided into naive T cells (TN), central memory cells (TCM), effector memory T cells (TEM), and regulatory T cells (Treg). The surface markers of each T lymphocyte subset will be described in detail below.

[0131] CD4+ T lymphocytes

[0132] CD4+ Treg cells: CD4+ CD25+ CD127-

[0133] CD4+ TN cells: CD4+CD45RA+CCR7+

[0134] CD4+TCM cells: CD4+ CD45RA-CCR7+

[0135] CD4+ TEM cells: CD4+ CD45RA-CCR7-

[0136] CD4+Th1 cells: CD4+ CD45RA-CXCR3+CCR6-

[0137] CD4+Th2 cells: CD4+ CD45RA-CXCR3-CCR6-

[0138] CD4+Th17 cells: CD4+ CD45RA-CXCR3-CCR6+

[0139] CD4+ Tfh cells: CD4+ CD45RA-PD-1+CXCR5+

[0140] CD8+ T lymphocytes

[0141] CD8+ Treg cells: CD8+ CD25+ CD127-

[0142] CD8+ TN cells: CD8+ CD45RA+ CCR7+

[0143] CD8+TCM cells: CD8+ CD45RA-CCR7+

[0144] CD8+ TEM cells: CD8+ CD45RA-CCR7-

[0145] Cells were resuspended in a surface marker mixture (antibody-conjugated fluorescent dye) in 96V bottom-plates, 1 × 10⁶ cells per well. 6 Cells were stained in a 50µL staining system (FACS buffer: 1×PBS 500ml, 0.2% BSA (1g), 2mM EDTA (0.5M EDTA 2ml), 0.1% sodium azide (10% sodium azide 5ml)) at room temperature in the dark for 30 minutes. Cells were then washed with 250µL BD Pharmaceutical Stain Buffer by centrifugation at 600g for 6 to 8 minutes, and the supernatant was discarded, leaving a volume no greater than 50µL.

[0146] The following operations are performed according to the experimental objectives: one method is to directly fix the cells, and the other is to culture and stimulate cells to fix them.

[0147] Method 1: Resuspend the cell clumps directly in 50µL of preheated BD phosflow Fix Buffer I and mix thoroughly using a pipette. Incubate the cells at 37°C for 10-12 minutes.

[0148] Method 2: Resuspend cells in complete culture medium (lymphocyte culture medium: 1640 + 10% FBS + 1mM sodium pyruvate + 1× penicillin / streptomycin + 1× non-essential amino acids + 50μM β-mercaptoethanol + 10mM HEPES) at a density of 1×10⁻⁶ cells / mL. 7 Cells / mL, 100µL was transferred to a 96V plate and treated with appropriate stimulation. Untreated control samples should be placed in parallel and incubated at 37°C for a suitable time (this application adds 100ng / mL hIL-2 (37°C water bath for 15 minutes), 20ng / mL hIL-6 (37°C water bath for 15 minutes), and 5ng / mL hIL-12 (37°C, 5% CO2 incubation for 15 hours)). Immediately after the stimulation period, an equal volume of preheated BD phosflow Fix Buffer I was added and mixed with a pipette (this application uses method two to fix cells).

[0149] Penetrating cell biomembranes: Divide the same sample into two equal parts, centrifuge to collect the cells at 600 g for 6 to 8 minutes, discard the supernatant, leaving a volume of less than 50 µL, and vortex to disperse the cell clumps; resuspend the cells in BD phosflow Perm Buffer III, 1 × 10⁻⁶ cells per aliquot. 7Cells, at least 500 µL; mix thoroughly with a multipipe strainer and incubate on ice in the dark for 30 minutes. Add 2 volumes of BD Pharmaceutical Stain Buffer, centrifuge at 600g for 6-8 minutes to wash cells, discard the supernatant, and leave a remaining volume of no more than 50 µL.

[0150] (3) Protein phosphorylation flow cytometry staining

[0151] The study detects phosphorylation levels of signal transduction and transcriptional activator families of T lymphocyte subsets, including pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins. A peer control is set up: an isotype control uses immunoglobulins of the same species, subtype, dose, and subtype as the primary antibody to eliminate background staining caused by non-specific antibody binding to cells. The isotype control is a true negative control; it can be used not only to set the voltage of the flow cytometer but also to eliminate the need for the expensive and cumbersome recombinant cytokine competitive blocking step.

[0152] Resuspend the cells in the prepared protein phosphorylation flow cytometry antibody mixture, and perform an isotype control for each sample, at a ratio of 1×102. 6 Cells were prepared in 50µL of staining system (FACS buffer: 1×PBS 500ml, 0.2% BSA (1g), 2 mM EDTA (0.5M EDTA 2ml), 0.1% sodium azide (10% sodium azide 5ml)). The mixture was thoroughly mixed using a multipipe strainer and incubated at 37°C in the dark for 30 minutes. Twice the volume of BD Pharmaceutical Stain Buffer was added, and the cells were centrifuged at 600g for 6-8 minutes. The supernatant was collected, with a remaining volume not exceeding 50µL. The cells were washed twice. The cells were resuspended in the staining system (FACS buffer: 1×PBS 500ml, 0.2% BSA (1g), 2 mM EDTA (0.5M EDTA 2ml), 0.1% sodium azide (10% sodium azide 5ml)) to a final concentration of 5-10×10⁻⁶ cells / mL. 6 Cells / mL, filtered through a 40µm filter, and 200µL of cell suspension is used for analysis.

[0153] (4) Flow cytometry data acquisition

[0154] Data were acquired using a BD LSRFortessa™ flow cytometer and FACS Diva 8.0 software. Compensation was created using 18 single-stained tubes before sample loading, and the BD LSRFortessa™ flow cytometer automatically calculated the compensation rules. Each sample was diluted to 5-10 × 10⁻⁶ before processing. 6 Cells / mL, maximum flow rate recorded 500,000 lymphocyte events.

[0155] (5) Flow cytometry data analysis

[0156] Data were analyzed using FlowJo_V10. The graphs show fluorescence intensity (MFI) on a logarithmic (double exponential) axis, with negative events displayed below the axis. One-dimensional plots show phosphorylation levels in cell subsets; two-dimensional plots show the stepwise logarithmic structure and cell subsets. Statistical significance was assessed using a t-test.

[0157] (6) Gating strategies for identifying various subsets of T lymphocytes

[0158] The controls are set as follows: a blank control without flow cytometry staining, a control for cell population clustering only stained on the surface, and an isotype control for protein phosphorylation flow cytometry staining. Select FSC for the X-axis and FSH for the Y-axis (or SSC for the X-axis and SSH for the Y-axis) to remove double adhesions and cell debris; remove dead cells using Zombie Aquarium. Then, select a gating tool (rectangular gate, elliptical gate, polygonal gate, or automatic gate) and select the lymphocyte population in the 2D point plot. Use the polygonal gate, rectangular gate, or cross gate tools to set gates and delineate cell subpopulations based on cell distribution density. Select a region gate to set the positive percentage of the protein phosphorylation flow cytometry 1D plot.

[0159] 3. Experimental Results

[0160] Figure 1 This diagram illustrates the phylogenetic model of T lymphocytes stained with a 17-color reagent set. 1A shows the lymphocyte population after excluding double adhesions, debris, and dead cells; 1B shows the phylogenetic model and cell subset differentiation after staining the surface of CD4+ T lymphocytes; 1C shows the phylogenetic model and cell subset differentiation after staining the surface of CD8+ T lymphocytes.

[0161] Figure 2 The effects of changes in the order of staining, fixation, and permeabilization on cell surface markers and protein phosphorylation levels are shown in 2A; the order of staining, fixation, and permeabilization is shown in 2B.

[0162] Figure 3The expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway of CD4+ T lymphocytes were detected by flow cytometry using protein phosphorylation (significant changes are indicated by %), with gray representing isotype controls and colored representing phosphorylation. 3A shows the expression level of pSTAT1 in CD4+ T lymphocytes; 3B shows the expression level of pSTAT3 in CD4+ T lymphocytes; 3C shows the expression level of pSTAT4 in CD4+ T lymphocytes; 3D shows the expression level of pSTAT5 in CD4+ T lymphocytes; and 3E shows the expression level of pSTAT6 in CD4+ T lymphocytes.

[0163] Figure 4 The expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway of CD8+ T lymphocytes were detected by flow cytometry (significant changes are indicated by %), gray represents isotype control, and colored represents phosphorylation.

[0164] This application experimentally demonstrates that, using the method described herein and following the surface-fixation-permeabilization treatment process, the phosphorylated proteins in peripheral blood mononuclear cells remain stable. This experiment detected STAT3 expression in CD4+ T cells and CD4+ TN cells, and STAT1 expression in CD4+ TEM cells and CD4+ Th17 cells. This experiment effectively detects the proportion of cell subsets and the phosphorylation levels of proteins in each cell subset.

[0165] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A method for simultaneously identifying cell subsets and phosphorylation levels of related factors in a sample, characterized in that, The method includes: identifying cell subpopulations using a first antibody mixture, and then detecting phosphorylation levels of related factors using a second antibody mixture, wherein the first antibody mixture consists of antibodies that specifically recognize cell surface markers, such as CD183, TCR αβ, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, and CD197; and the second antibody mixture consists of antibodies that specifically recognize phosphorylation sites of related factors, such as STAT1, STAT3, STAT4, STAT5, and STAT6. The antibody-coupled fluorescent dye in the first antibody mixture is composed of BUV496, BUV395, BUV737, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, Alexa Fluor 700 and APC-Cy7. The second antibody mixture contains an antibody-conjugated fluorescent dye, which is composed of AF488, BV421, APC, PECy7, and PerCP-Cy5.5; The cells were T lymphocytes, and the sample was PBMCs; Identification of cell subpopulations using a first antibody mixture also includes pre-staining cell fixation; Cells were fixed using BD phosflow Fix Buffer I; The method also includes incubating cells with interleukin before fixing the cells; The interleukin mentioned is human interleukin; The human interleukins include IL-2, IL-6 and IL-12; The concentration ratio of IL-2, IL-6 and IL-12 was 20:4:1; The method also includes penetrating the cell biomembrane after fixing the cells; The method for penetrating cell biomembranes involves centrifuging, precipitating, resuspending, and washing the cells. Cells were resuspended using BD phosflow Perm Buffer III; The method also includes analyzing the processed samples using flow cytometry.

2. The method according to claim 1, characterized in that, The ratio of antibodies conjugated with fluorescent dyes is as follows: CD183BUV395: TCR αβ BUV737: CD8a BV510: CD127 BV605: CD45RA BV650: CD279 BV711: CCR6 PE: CD185 PE / Dazzle 594: CD25 PECy5: CD4 Alexa Fluor 700: CD197 APC-Cy7 = 1:1:1:2:2:1:1:1:1:1:

2.

3. The method according to claim 1, characterized in that, The ratio of antibodies conjugated with fluorescent dyes is: STAT1AF488:STAT3BV421:STAT4APC:STAT5PECy7:STAT6PerCP-Cy5.5 = 1:1:1:1:

1.

4. The method according to claim 1, characterized in that, The staining system used to identify cell subpopulations using the first antibody mixture includes PBS, BSA, EDTA, and sodium azide.

5. The method according to claim 4, characterized in that, The staining system consisted of 1×PBS, 0.2% BSA (by volume), 2mM EDTA, and 0.1% sodium azide (by volume).

6. The method according to claim 4, characterized in that, The amount of the staining system used is 1×10 6 50 μL per cell.

7. The method according to claim 1, characterized in that, In the method of penetrating cell biomembranes, the cells were washed with BD Pharmaceutical Stain Buffer after centrifugation, precipitation, resuspension, and washing.

8. The method according to claim 1, characterized in that, The staining system used to identify cell subpopulations using a mixture of second antibodies includes PBS, BSA, EDTA, and sodium azide.

9. The method according to claim 8, characterized in that, The method also includes washing and resuspending the cells after staining.

10. The method according to claim 9, characterized in that, Cells were washed with Pharmingen Stain Buffer.

11. The method according to claim 9, characterized in that, Cells were resuspended using PBS, BSA, EDTA, and sodium azide.

12. The method according to claim 11, characterized in that, The concentrations of PBS, BSA, EDTA, and sodium azide are 1×PBS, 0.2% BSA (by volume), 2mM EDTA, and 0.1% sodium azide (by volume).

13. The method according to claim 1, characterized in that, The flow cytometer is a multicolor flow cytometer.

14. The use of a first antibody mixture and a second antibody mixture in the preparation of reagents for identifying cell subsets and phosphorylation levels of related factors in a sample, characterized in that, The first antibody mixture consists of antibodies that can specifically recognize cell surface markers, namely CD183, TCR αβ, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, and CD197; the second antibody mixture consists of antibodies that can specifically recognize phosphorylation sites of related factors, namely STAT1, STAT3, STAT4, STAT5, and STAT6. The antibody-conjugated fluorescent dye in the first antibody mixture and the second antibody mixture; The antibody-conjugated fluorescent dyes in the first antibody mixture consist of BUV496, BUV395, BUV737, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, Alexa Fluor 700, and APC-Cy7. The antibody-conjugated fluorescent dyes in the second antibody mixture consist of AF488, BV421, APC, PECy7, and PerCP-Cy5.5; The cells were T lymphocytes, and the sample was PBMCs; The reagent also includes interleukin; The interleukin mentioned is human interleukin; The human interleukins include IL-2, IL-6 and IL-12; The concentration ratio of IL-2, IL-6 and IL-12 was 20:4:1; The reagents also include BD phosflow Fix Buffer I, BD phosflow Perm Buffer III, and BDPharmingen Stain Buffer.

15. The application according to claim 14, characterized in that, The ratio of antibodies conjugated with fluorescent dyes is as follows: CD183BUV395: TCR αβ BUV737: CD8a BV510: CD127 BV605: CD45RA BV650: CD279 BV711: CCR6 PE: CD185 PE / Dazzle 594: CD25 PECy5: CD4 Alexa Fluor 700: CD197 APC-Cy7 = 1:1:1:2:2:1:1:1:1:1:

2.

16. The application according to claim 14, characterized in that, The ratio of antibodies conjugated with fluorescent dyes is: STAT1AF488:STAT3BV421:STAT4APC:STAT5PECy7:STAT6PerCP-Cy5.5 = 1:1:1:1:

1.

17. The application according to claim 14, characterized in that, The reagents also include a staining system.

18. The application according to claim 17, characterized in that, The staining system includes PBS, BSA, EDTA, and sodium azide.

19. The application according to claim 18, characterized in that, The staining system consisted of 1×PBS, 0.2% BSA (by volume), 2mM EDTA, and 0.1% sodium azide (by volume).

20. The use of a first antibody mixture and a second antibody mixture in the preparation of a kit for simultaneously identifying cell subsets and phosphorylation levels of related factors in a sample; characterized in that, The first antibody mixture consists of antibodies that can specifically recognize cell surface markers, namely CD183, TCR αβ, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, and CD197; the second antibody mixture consists of antibodies that can specifically recognize phosphorylation sites of related factors, namely STAT1, STAT3, STAT4, STAT5, and STAT6. The antibody-conjugated fluorescent dye in the first antibody mixture and the second antibody mixture; The antibody-conjugated fluorescent dyes in the first antibody mixture consist of BUV496, BUV395, BUV737, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, Alexa Fluor 700, and APC-Cy7. The antibody-conjugated fluorescent dyes in the second antibody mixture consist of AF488, BV421, APC, PECy7, and PerCP-Cy5.5; The cells were T lymphocytes, and the sample was PBMCs; The kit also includes interleukin; The interleukin mentioned is human interleukin; The human interleukins include IL-2, IL-6 and IL-12; The concentration ratio of IL-2, IL-6 and IL-12 was 20:4:1; The kit also includes BD phosflow Fix Buffer I, BD phosflow Perm Buffer III, and BDPharmingen Stain Buffer.

21. The application according to claim 20, characterized in that, The ratio of antibodies conjugated with fluorescent dyes is as follows: CD183BUV395: TCR αβ BUV737: CD8a BV510: CD127 BV605: CD45RA BV650: CD279 BV711: CCR6 PE: CD185 PE / Dazzle 594: CD25 PECy5: CD4 Alexa Fluor 700: CD197 APC-Cy7 = 1:1:1:2:2:1:1:1:1:1:

2.

22. The application according to claim 20, characterized in that, The ratio of antibodies conjugated with fluorescent dyes is: STAT1AF488:STAT3BV421:STAT4APC:STAT5PECy7:STAT6PerCP-Cy5.5 = 1:1:1:1:

1.

23. The application according to claim 20, characterized in that, The kit also includes a staining system.

24. The application according to claim 23, characterized in that, The staining system includes PBS, BSA, EDTA, and sodium azide.

25. The application according to claim 24, characterized in that, The staining system consisted of 1×PBS, 0.2% BSA (by volume), 2mM EDTA, and 0.1% sodium azide (by volume).

26. The application according to claim 20, characterized in that, The kit also includes packaging materials for packaging the composition.

27. The application according to claim 26, characterized in that, The kit also includes instructions.

28. A system for simultaneously identifying cell subsets and phosphorylation levels of related factors in a sample, characterized in that, The system includes: A memory that stores processor-readable instructions; and one or more processors arranged to read and execute the instructions stored in the memory; Wherein, the processor-readable instructions include instructions arranged to control the computer to perform the method of any one of claims 1-13; The cells in question are T lymphocytes.

29. The use of the method of any one of claims 1-13 or the system of claim 28 in identifying cell subsets and phosphorylation levels of related factors in a sample; wherein the cells are T lymphocytes.

Images