Method for simultaneously identifying phosphorylation levels of cell subpopulation and related factors

Abstract

The invention discloses a method for simultaneously identifying phosphorylation levels of cell subgroups and related factors. According to the method, the protein phosphorylation flow cytometry staining step is simplified, the staining effect is not affected, 17 kinds of stained cells can be detected at the same time, in addition, the cost is saved, and higher efficiency is achieved. According to the method provided by the invention, more accurate distinguishing capability can be provided, more information can be obtained from a few-cell specimen, abnormal cells can be detected at a low level, the laboratory efficiency is improved, and the method is particularly suitable for research of an immune system and has a wide prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for simultaneously identifying cell subpopulations and phosphorylation levels of related factors. Background Art

[0002] Protein phosphorylation is the process of transferring a phosphate group to a substrate protein under the catalysis of a kinase. Phosphorylation primarily occurs at tyrosine, serine, and threonine residues in substrate proteins, causing conformational changes in the protein. This in turn dynamically regulates protease activity and protein-protein interactions, leading to a sequential protein phosphorylation cascade that rapidly transmits signals and ultimately regulates various events closely related to life, such as cellular 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 the protein phosphorylation process and signaling pathways is crucial for further studying its role in disease.

[0003] Traditionally, immunoblotting is commonly used to detect the phosphorylation levels of molecules in the JAK-STAT pathway, but the operation time is long and the steps are complicated, which is not conducive to the rapid, accurate and quantitative detection of protein expression levels; and it cannot simultaneously detect the phosphorylation levels of molecules in the JAK-STAT pathway in various cell subpopulations.

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

[0005] To overcome the deficiencies of the prior art, the present invention provides a method for simultaneously identifying cell subpopulations and the phosphorylation levels of related factors.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

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

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

[0009] Furthermore, the usage ratio of the antibodies conjugated with fluorescent dyes is: 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 antibodies in the second antibody mixture are coupled to fluorescent dyes, and the fluorescent dyes include one or more of AF488, BV421, APC, PECy7, and PerCP-Cy5.5.

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

[0012] Furthermore, the primary antibody mixture was used to identify cell subpopulations using a staining system including PBS, BSA, EDTA, and sodium azide.

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

[0014] Furthermore, the amount of the dyeing system is per 1×10 6 50 μL for each cell.

[0015] Furthermore, identifying cell subpopulations using the primary antibody cocktail further comprises fixing the cells before staining.

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

[0017] Furthermore, the method further comprises incubating the cells with interleukin before fixing the cells.

[0018] Furthermore, the interleukin is 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 was 20:4:1.

[0021] Furthermore, the method further comprises penetrating the cell biomembrane after fixing the cells.

[0022] Furthermore, the method for penetrating the cell biomembrane is to resuspend and wash the cells after centrifugation.

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

[0024] Furthermore, the cells were washed with BD Pharmingen Stain Buffer.

[0025] Furthermore, a secondary antibody mixture was used to identify cell subpopulations using a staining system including PBS, BSA, EDTA, and sodium azide.

[0026] Furthermore, the method further comprises washing the cells and resuspending the cells after staining.

[0027] Furthermore, the cells were washed with Pharmingen Stain Buffer.

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

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

[0030] Furthermore, the cells are T lymphocytes.

[0031] Furthermore, the sample is PBMC.

[0032] Furthermore, the method further comprises analyzing the processed sample using a flow cytometer.

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

[0034] The second aspect of the present invention provides a reagent for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, wherein the reagent comprises: 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 antibodies in the first antibody mixture and the second antibody mixture are coupled with fluorescent dyes.

[0036] 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.

[0037] Furthermore, the usage ratio of the antibodies conjugated with fluorescent dyes is: 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-coupled fluorescent dye in the second antibody mixture includes one or more of AF488, BV421, APC, PECy7, and PerCP-Cy5.5.

[0039] Furthermore, the usage ratio of the antibodies coupled 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 dyeing system.

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

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

[0043] Furthermore, the reagent also includes interleukin.

[0044] Furthermore, the interleukin is 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 was 20:4:1.

[0047] Furthermore, the reagents further 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 PBMC.

[0050] The third aspect of the present invention provides a kit for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, wherein the kit comprises 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 subpopulations and phosphorylation levels of related factors in a sample, the system comprising:

[0055] a memory storing 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 configured to control a computer to execute 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 use 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 related 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 cytometric staining steps without affecting the staining effect, and can simultaneously detect 17 types of stained cells. In addition, it also saves costs and is more efficient. The method provided by this application can provide more precise differentiation capabilities, can obtain more information from small cell specimens, can detect abnormal cells at low levels, and increase laboratory efficiency. It is particularly suitable for research on the immune system and has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Figure 1 is a schematic diagram of the gating strategy for T lymphocytes stained with a 17-color reagent set. Figure 1A shows the lymphocyte population after excluding doublets, debris, and dead cells. Figure 1B shows the gating strategy and differentiation of cell subsets after surface staining of CD4+ T lymphocytes in T lymphocytes. Figure 1C shows the gating strategy and differentiation of cell subsets after surface staining of CD8+ T lymphocytes in T lymphocytes.

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

[0065] Figure 3 Figure 3A shows the expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway in CD4+ T lymphocytes detected by protein phosphorylation flow cytometry (significant changes are indicated by %, gray represents isotype control, and color represents phosphorylation). Figure 3A shows the expression level of pSTAT1 in CD4+ T lymphocytes, Figure 3B shows the expression level of pSTAT3 in CD4+ T lymphocytes, Figure 3C shows the expression level of pSTAT4 in CD4+ T lymphocytes, Figure 3D shows the expression level of pSTAT5 in CD4+ T lymphocytes, and Figure 3E shows the expression level of pSTAT6 in CD4+ T lymphocytes.

[0066] Figure 4This is a graph using protein phosphorylation flow cytometry to detect the expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway of CD8+ T lymphocytes (significant changes are indicated by %, gray represents isotype control, and color represents phosphorylation). DETAILED DESCRIPTION

[0067] The following provides definitions of some terms used in this specification. Unless defined otherwise, 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 belongs.

[0068] The present invention provides a method for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample. The method comprises: using a first antibody mixture to identify cell subpopulations, and then using a second antibody mixture to detect the phosphorylation levels of related factors, 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.

[0069] In some embodiments, the methods of the present application can identify multiple specific cell types and simultaneously identify the phosphorylation levels of related factors. These cell types include T lymphocytes, which include CD4+ and CD8+ T cells, which are 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 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 the present application can simultaneously identify the surface markers of 17 cells and the levels of 5 phosphorylated molecules. The presence or absence of 17 cell surface markers and 5 phosphorylated molecules is determined simultaneously (i.e., at the same time). Utilizing the method of the present application, an analysis can be performed on a cell colony and the presence or absence of the 17 markers and 5 phosphorylated molecules can be determined thereby. It is not necessary to run more than one analysis or to analyze with multiple samples.

[0071] In some embodiments, antibodies that can specifically bind to various cell surface markers are known in the art. When an antibody binds preferentially or with high affinity to a certain cell surface marker sequence but does not substantially bind, does not bind, or binds only with low affinity to other cell surface markers or other proteins, then the antibody specifically binds to the cell surface marker sequence.

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

[0073] In some embodiments, each of the fluorescently labeled antibodies in the present application is typically titrated to an appropriate concentration for use in the methods of the present application, and each antibody is typically titrated at 1:10 to 1:1000000, for example, 1:50, 1:100, 1:200, 1:300, 1:500, 1:1000, 1:10,000, 1:50,000, 1:100,000.

[0074] In some embodiments, the cell population is typically present in a 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 the present application, the sample is PBMC. Freshly prepared PBMC are generally used. If frozen PBMC are used, the conditions for cell recovery after thawing should be optimized to ensure appropriate basal phosphorylation levels in unstimulated cells and appropriate cellular responses to stimulation.

[0075] Samples are typically processed prior to testing, for example by centrifugation or by passing them through a membrane that filters out unwanted molecules or cells (e.g., red blood cells). Samples can be assayed immediately after collection. Samples are also typically stored prior to testing, preferably at a temperature below -70°C.

[0076] The present 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 test kit can be packaged in the form of an aqueous medium or in a lyophilized form. Suitable containers in the test kit generally include at least a vial, test tube, flask, plastic bottle, syringe or other container, in which a component can be placed, and preferably, can be appropriately divided. When there is more than one component in the test kit, the test kit will generally also include a second, third or other additional container, in which additional components are placed separately. However, the components of different combinations can be contained in a vial. The test kit of the present invention will generally also include a container for accommodating reactants, sealed for commercial sale. Such a container can include an injection molded or blow molded plastic container, in which the required vial can be retained.

[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 test kit of the present application may additionally include one or more other reagents or instruments that enable any of the embodiments mentioned above to be performed. The reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), an apparatus for obtaining a sample from a subject (e.g., a container or an instrument including a needle) and / or other reagents required for FACS analysis. The reagent may be present in the test kit in a dry state so that the reagent can be resuspended with a liquid sample. The test kit may also optionally include instructions for enabling the test kit to be used in the method of the present application or details on which sample the method can be used for.

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

[0081] a memory storing processor-readable instructions; and

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

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

[0084] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0085] Example

[0086] 1. Experimental Materials

[0087] The fluorescently labeled antibodies used in this application are specifically binding monoclonal antibodies. The name, manufacturer, clone number, and antibody titer of the fluorescent antibodies are shown in Table 1. The surface marker antibody titer is determined based on whether the cell surface marker binds firmly to fluorescein and whether the fluorescently labeled antibody to the cell surface marker can tolerate fixatives and permeabilization agents.

[0088] Table 1 Antibodies

[0089] PBMC: Derived from rheumatoid arthritis patients collected in Beijing Hospital in May 2025.

[0090] Instruments and equipment:

[0091] BD LSRFortessa™ flow cytometer (BD, Cat. No. 647800)

[0092] Centrifuge 5801 R centrifuge (Eppendorf, Germany)

[0093] Centrifuge 5417 R centrifuge (Eppendorf, Germany)

[0094] Manual defrost refrigerator (Haier Company)

[0095] -80°C ultra-low temperature freezer (Thermo Scientific)

[0096] 37 degrees Celsius water bath

[0097] 30-300µL multichannel pipette (Eppendorf)

[0098] 1-10µL pipette (Eppendorf)

[0099] 100µL pipette (Eppendorf)

[0100] 1000µL pipette (Eppendorf)

[0101] Reagents and consumables:

[0102] 5 ml round-bottom polystyrene flow tube (Falcon, catalog number: 352008)

[0103] 40µm blue sterile cell strainer (Falcon, Cat. No. 35234)

[0104] 50 ml sterile centrifuge tube (Sorfa, catalog number: T124-4)

[0105] 15 ml centrifuge tube (ServiceBio, catalog number: ep-1500)

[0106] 1.5 ml centrifuge tube (ServiceBio, catalog number: ep-150)

[0107] 2 ml centrifuge tube (ServiceBio, catalog number: ep-200)

[0108] 200µL centrifuge tube (Axygen, catalog number: pcr-02-c)

[0109] Transparent polystyrene 96V bottom well plate 3897 (Corning, Cat. No. 07-200-108)

[0110] Lymphocyte separation medium, 500 ml (Alere Technologies AS, catalog number: AXS-1114546)

[0111] Dimethyl sulfoxide (Sigma-Aldrich, product number: D2650-100ML)

[0112] Fetal bovine serum, 500 ml (Sciencell, Cat. No. 0500)

[0113] RPMI 1640 culture medium (Gibco, catalog number: 11875093)

[0114] Cytostimulatory factors

[0115] Phosphate buffered saline (1× PBS) (HyClone, catalog number: SH30256.01)

[0116] BD phosflow Perm Buffer III (Cat. No. 558050)

[0117] BD phosflow Fix Buffer I (Cat. No. 557870)

[0118] BD Pharmingen Stain Buffer (FBS) (Cat. No. 554656)

[0119] Fluorescent protein-conjugated antibodies (BD, Biolegend, eBioscience)

[0120] Reagent preparation

[0121] (1) Incubate BD phosflow Fix Buffer I in a 37°C water bath and BD phosflow PermBuffer III in a -20°C refrigerator.

[0122] (2) RPMI 1640 medium was prepared into a complete medium containing 10% FBS.

[0123] (3) Red blood cell lysis solution: Potassium bicarbonate (KHCO3) 1.0g, ammonium chloride (NH4CL) 8.3g, EDTA-Na2 0.037g, add dihydrogen phosphate (H2O) to 1000ml to prepare the working solution.

[0124] 2. Experimental methods

[0125] (1) Preparation of human peripheral blood mononuclear cells (PBMC)

[0126] After removing the plasma and anticoagulating venous blood, dilute it at a volume ratio of 1:1 blood to 1× PBS. Inject the diluted blood onto the lymphocyte separation medium at a ratio of 2:1 blood to lymphocyte separation medium. Centrifuge at 3 units of speed (upper limit) and 2 units of speed (lower limit) at 500g for 30 minutes at 20°C. Aspirate the white, flocculent mononuclear cells, resuspend them in 4 volumes of RPMI-1640, and wash them twice by centrifugation at 600g for 6 minutes. If the aspirated mononuclear cells contain a high number of red blood cells, wash them by centrifuging 10 mL of peripheral blood with 1 mL of red blood cell lysis buffer for 3 minutes at room temperature, then centrifuge them at a volume of 5× PBS.

[0127] (2) Surface protein flow cytometry staining

[0128] T lymphocyte subsets are identified based on cell surface markers. T lymphocytes include CD4+ and CD8+ T cells, which are 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 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 surface markers of each T lymphocyte subset are described in detail below.

[0129] CD4+ T lymphocytes

[0130] CD4+Treg cells: CD4+ CD25+CD127-

[0131] CD4+TN cells: CD4+CD45RA+CCR7+

[0132] CD4+ TCM cells: CD4+ CD45RA-CCR7+

[0133] CD4+TEM cells: CD4+ CD45RA-CCR7-

[0134] CD4+ Th1 cells: CD4+ CD45RA-CXCR3+CCR6-

[0135] CD4+ Th2 cells: CD4+ CD45RA-CXCR3-CCR6-

[0136] CD4+ Th17 cells: CD4+ CD45RA-CXCR3-CCR6+

[0137] CD4+Tfh cells: CD4+ CD45RA-PD-1+CXCR5+

[0138] CD8+ T lymphocytes

[0139] CD8+Treg cells: CD8+ CD25+CD127-

[0140] CD8+TN cells: CD8+ CD45RA+CCR7+

[0141] CD8+TCM cells: CD8+ CD45RA-CCR7+

[0142] CD8+TEM cells: CD8+ CD45RA-CCR7-

[0143] Resuspend the cells in a 96V-bottom plate with a surface marker mixture (antibody-conjugated fluorescent dye) at a rate of 1 × 10 6 For each cell, stain with 50 µL of FACS buffer (1× PBS (500 mL), 0.2% BSA (1 g), 2 mM EDTA (2 mL), 0.1% sodium azide (5 mL)) at room temperature in the dark for 30 minutes. Add 250 µL of BD Pharmingen Stain Buffer and wash the cells by centrifugation at 600 g for 6 to 8 minutes. Remove the supernatant, leaving a residual volume of no more than 50 µL.

[0144] The following operations are performed according to the experimental purpose. One method is to directly fix the cells, and the other is to culture the stimulated cells and then fix the cells.

[0145] Method 1: Directly resuspend the cell pellet in 50 µL of pre-warmed BD phosflow Fix Buffer I and mix thoroughly by pipetting. Incubate the cells at 37°C for 10-12 minutes.

[0146] Method 2: Resuspend the cells in complete 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 7 For each cell / mL, transfer 100µL to a 96V-bottom plate and treat the cells with the appropriate stimulus. Place an untreated control sample in parallel and incubate at 37°C for an appropriate period of time (this application includes 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 for 15 hours). Immediately after the stimulation period, add an equal volume of prewarmed BD phosflow Fix Buffer I and mix thoroughly with a pipette (this application uses Method 2 for cell fixation).

[0147] To penetrate the cell membrane: Divide the sample into two equal parts, centrifuge the cells at 600 g for 6 to 8 minutes, remove the supernatant, and vortex to disperse the cell clumps when the remaining volume is less than 50 µL; resuspend the cells in BD phosflow Perm Buffer III at 1 × 10 7Cells, at least 500 μL; mix thoroughly by pipetting and incubate on ice for 30 minutes in the dark. Add 2 volumes of BD PharmingenStain Buffer and centrifuge at 600 g for 6-8 minutes to wash the cells. Discard the supernatant, leaving no more than 50 μL.

[0148] (3) Protein phosphorylation flow cytometry staining

[0149] Detects phosphorylation levels of signal transducers and activators of transcription (STARs) in T lymphocyte subsets, including pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins. Isotype controls: Isotype controls are immunoglobulins of the same species, subtype, dose, and subtype as the primary antibody to eliminate background staining caused by nonspecific antibody binding to cells. Isotype controls are true negative controls that not only set flow cytometer voltages but also eliminate the need for expensive and cumbersome recombinant cytokine competitive blocking steps.

[0150] Resuspend the cells with the prepared protein phosphorylation flow cytometry antibody mixture. At the same time, an isotype control should be made for each sample. 6 For each cell, add 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)). Mix well by pipetting and incubate at 37 degrees Celsius in a dark incubator for 30 minutes. Add 2 volumes of BD Pharmingen Stain Buffer, centrifuge at 600g for 6-8 minutes, remove the supernatant, and leave a volume of no more than 50µL. Wash twice more. Resuspend the cells in 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, filtered through a 40µm filter, and 200µL of the cell suspension was tested on the instrument.

[0151] (4) Flow cytometry data acquisition

[0152] Data were collected using a BD LSRFortessa™ flow cytometer and FACS Diva 8.0 software. Compensation was created using 18 single-stained tubes before loading, and the BD LSRFortessa™ flow cytometer automatically calculated the compensation rule. Each sample was diluted to 5-10×10 before loading. 6 cells / ml, and 500,000 lymphocyte events were recorded at the highest flow rate.

[0153] (5) Flow cytometry data analysis

[0154] Data were analyzed using FlowJo v10. Graphs display fluorescence intensity (MFI) on a logarithmic (biexponential) axis, with negative events plotted below the axis. One-dimensional plots display phosphorylation levels in cell subpopulations, while two-dimensional plots display stepwise logistic regression and cell subpopulations. Statistical significance was assessed using a Student's Student T-test.

[0155] (6) Gating strategies for identifying T lymphocyte subsets

[0156] Controls included a blank control (no flow cytometry staining), a position control for cell populations stained only on the surface, and an isotype control for protein phosphorylation flow cytometry. Select FSC on the X-axis and FSH on the Y-axis (or SSC on the X-axis and SSH on the Y-axis) to remove doublets and cell debris; use ZombieAquarium to remove dead cells. Then, select a gating tool (rectangular, elliptical, polygonal, or automatic) to select the lymphocyte population in the 2D dot plot. Use the polygonal, rectangular, and cross gate tools to gate and delineate cell subpopulations based on cell density. Select a regional gate to set the positive percentage for the 1D protein phosphorylation flow cytometry plot.

[0157] 3. Experimental results

[0158] Figure 1 Diagrams showing the gating strategy for T lymphocytes stained with the 17-color panel. 1A shows the lymphocyte population after excluding doublets, debris, and dead cells; 1B shows the gating strategy and cell subset differentiation after surface staining of CD4+ T lymphocytes; 1C shows the gating strategy and cell subset differentiation after surface staining of CD8+ T lymphocytes.

[0159] Figure 2 The effects of changing the order of staining, fixation, and permeabilization on cell surface markers and protein phosphorylation levels are demonstrated; 2A uses the order of staining for surface markers, fixation, and permeabilization; 2B uses the order of fixation, staining for surface markers, and permeabilization.

[0160] Figure 3Protein phosphorylation flow cytometry was used to assess the expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway in CD4+ T lymphocytes (significant changes are indicated by percentage; gray represents isotype control, and color represents 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.

[0161] Figure 4 Protein phosphorylation flow cytometry was used to detect the expression levels of pSTAT1, pSTAT3, pSTAT4, pSTAT5, and pSTAT6 proteins in the JAK-STAT pathway of CD8+ T lymphocytes (significant changes are indicated by %, gray represents isotype control, and color represents phosphorylation).

[0162] This application demonstrates through experiments that, using the present method and following the surface-fixation-permeabilization treatment, the phosphorylated proteins of 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 is highly capable of detecting the proportion of cell subsets and the protein phosphorylation levels of each cell subset.

[0163] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A method for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, characterized in that: The method includes: using a first antibody mixture to identify cell subpopulations, and then using a second antibody mixture to detect the phosphorylation level of related factors, wherein the antibodies in the first antibody mixture include: one or more of CD183, CD8a, CD127, CD45RA, CD279, CCR6, CD185, CD25, CD4, CD197, and the antibodies in the second antibody mixture include: one or more of STAT1, STAT3, STAT4, STAT5, STAT6.

2. The method according to claim 1, characterized in that The antibodies in the first antibody mixture are coupled to fluorescent dyes, wherein the fluorescent dyes include one or more of BUV395, BV510, BV605, BV650, BV711, PE, PE / Dazzle 594, PECy5, Alexa Fluor 700, and APC-Cy7; Preferably, the usage ratio of the antibodies conjugated with fluorescent dyes is: CD183 BUV395:CD8a BV510:CD127 BV605:CD45RA BV650:CD279 BV711:CCR6 PE:CD185 PE / Dazzle 594:CD25 PECy5:CD197 APC-Cy7=1:1:2:2:1:1:1:1:

2.

3. The method according to claim 1, characterized in that The antibodies in the second antibody mixture are coupled to fluorescent dyes, wherein the fluorescent dyes include one or more of AF488, BV421, APC, PECy7, and PerCP-Cy5.5; Preferably, the usage ratio of the antibodies coupled with fluorescent dyes is: STAT1 AF488: STAT3 BV421: STAT4 APC: STAT5 PECy7: STAT6 PerCP-Cy5.5=1:1:1:1:

1.

4. The method according to claim 1, wherein The staining system used to identify cell subpopulations using a mixture of primary antibodies included PBS, BSA, EDTA, and sodium azide; Preferably, the concentration of the staining system is 1×PBS, 0.2% BSA, 2mM EDTA, 0.1% sodium azide; Preferably, the amount of the dyeing system is per 1×10 6 50 μL for each cell.

5. The method according to claim 1, wherein Identification of cell subpopulations using a cocktail of primary antibodies also involves fixing cells before staining; Preferably, cells are fixed using BD phosflow Fix Buffer I; Preferably, the method further comprises incubating the cells with interleukin before fixing the cells; Preferably, the interleukin is a human interleukin; Preferably, the human interleukins include IL-2, IL-6, and IL-12; Preferably, the concentration ratio of IL-2, IL-6, and IL-12 is 20:4:1; Preferably, the method further comprises penetrating the cell biomembrane after fixing the cells; Preferably, the method of penetrating the cell biomembrane is to resuspend and wash the cells after centrifugation; Preferably, resuspend the cells in BD phosflow Perm Buffer III; Preferably, cells are washed using BD Pharmingen Stain Buffer.

6. The method according to claim 1, characterized in that Identification of cell subpopulations using a secondary antibody cocktail The staining system used included PBS, BSA, EDTA, and sodium azide; Preferably, the method further comprises washing the cells and resuspending the cells after staining; Preferably, cells are washed using Pharmingen Stain Buffer; Preferably, cells are resuspended using PBS, BSA, EDTA, or sodium azide; Preferably, the concentrations of PBS, BSA, EDTA, and sodium azide are 1×PBS, 0.2% BSA, 2 mM EDTA, and 0.1% sodium azide, respectively; Preferably, the cells are T lymphocytes; Preferably, the sample is PBMC; Preferably, the method further comprises analyzing the processed sample using a flow cytometer; Preferably, the flow cytometer is a multicolor flow cytometer.

7. A reagent for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, characterized in that: The reagents include: 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; Preferably, the antibodies in the first antibody mixture and the second antibody mixture are conjugated with fluorescent dyes; Preferably, 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; Preferably, the usage ratio of the antibodies conjugated with fluorescent dyes is: CD183 BUV395:CD8a BV510:CD127 BV605:CD45RA BV650:CD279 BV711:CCR6 PE:CD185 PE / Dazzle 594:CD25 PECy5:CD197 APC-Cy7=1:1:2:2:1:1:1:1:2; Preferably, the antibody-conjugated fluorescent dye in the second antibody mixture includes one or more of AF488, BV421, APC, PECy7, and PerCP-Cy5.5; Preferably, the usage ratio of the fluorescent dye-coupled antibodies is: STAT1 AF488: STAT3 BV421: STAT4 APC: STAT5 PECy7: STAT6 PerCP-Cy5.5 = 1:1:1:1:1; Preferably, the reagent further comprises a staining system; Preferably, the staining system comprises PBS, BSA, EDTA, and sodium azide; Preferably, the concentration of the staining system is 1×PBS, 0.2% BSA, 2mM EDTA, 0.1% sodium azide; Preferably, the agent further comprises interleukin; Preferably, the interleukin is a human interleukin; Preferably, the human interleukins include IL-2, IL-6, and IL-12; Preferably, the concentration ratio of IL-2, IL-6, and IL-12 is 20:4:1; Preferably, the reagent further comprises one or more of BD phosflow Fix Buffer I, BD phosflow Perm Buffer III, and BD Pharmingen Stain Buffer; Preferably, the cells are T lymphocytes; Preferably, the sample is PBMC.

8. A kit for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, characterized in that: The kit comprises the reagent according to claim 7; Preferably, the cells are T lymphocytes; Preferably, the kit further comprises packaging materials for packaging the composition; Preferably, the kit further comprises instructions.

9. A system for simultaneously identifying cell subpopulations and phosphorylation levels of related factors in a sample, characterized in that: The system comprises: a memory storing 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 a computer to execute the method according to any one of claims 1 to 6; Preferably, the cells are T lymphocytes.

10. Use of the method according to any one of claims 1 to 6, the reagent according to claim 7, the kit according to claim 8, or the system according to claim 9 in identifying cell subpopulations and phosphorylation levels of related factors in a sample; Preferably, the related factors include one or more of STAT1, STAT3, STAT4, STAT5, and STAT6; Preferably, the cells are T lymphocytes.

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