Immune cell sorting method

Abstract

The invention belongs to the technical field of biology, particularly relates to an immune cell sorting method, and more particularly relates to a peripheral blood immune cell subtype sorting method. By using the method disclosed by the invention, a blood sample can be efficiently utilized, for example, only 4mL of blood is used, and at most 24 different immune cell subtypes can be sorted, so that the method is used for monitoring or inspecting immune cells in clinical peripheral blood and a cell multiomics analysis process in the scientific research field.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, relates to a method for sorting immune cells, and more particularly, to a method for sorting peripheral blood immune cell subtypes. Background Art

[0002] Immune cells are cells that make up the immune system. They play an important role in protecting the body from pathogens (such as bacteria, viruses, parasites) and foreign substances, removing damaged or cancerous cells, and maintaining the stability of the body's internal environment. The types of immune cells can be divided into two categories according to their functions and sources: innate immune cells and adaptive immune cells. These two major categories of immune cells can be divided into many cell subtypes. These immune cell subtypes each have their own functions in the immune system and play a specific role. Therefore, the typing and detection of immune cells provides important information for understanding and monitoring the functions of the immune system, and plays a key role in clinical diagnosis, treatment monitoring, disease research, and drug development. The typing and sorting of immune cells also provides research materials and experimental basis for the subsequent acquisition of multi-omics data and the multi-angle analysis of the mechanism of disease and drug action. It is an indispensable technical means in modern medicine and biological research. Only by sorting out accurate, high-purity, and rich immune cell subtypes can we better provide the best research materials for the pathogenicity and treatment mechanisms of subsequent diseases caused by abnormal immune systems.

[0003] Peripheral blood samples are the most commonly used and most easily accessible clinical resources in clinical pathology research. However, clinical detection of peripheral blood immune cells is limited to a few specific markers, and the detection range is not comprehensive enough. Only by testing or sorting as many immune cell types as possible to determine the multi-omics parameters of cells can the body's immune system be more comprehensively monitored and intervention measures be taken in a timely manner. Therefore, the simultaneous detection of multiple immune cell subtypes based on blood is also urgently needed for clinical testing.

[0004] Therefore, there is a need in the art for a method that can efficiently use blood samples to sort out as many immune cell subtypes as possible for use in the monitoring or testing of immune cells in clinical peripheral blood and in the multi-omics analysis of cells in the scientific research field. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a method for sorting immune cells, comprising sorting at least eight of the following immune cells according to markers:

[0006] Application of CD45 + CD3 + CD4 + CD25 + CD127 lowCombined sorting of regulatory T cells (Treg);

[0007] Application of CD45 + CD3 + CD4 + CCR6 - CCR4 - CXCR3 + Combined sorting of T4 Th1 cells;

[0008] Application of CD45 + CD3 + CD4 + CCR6 - CCR4 + CXCR3 - Combined sorting of T4 Th2 cells;

[0009] Application of CD45 + CD3 + CD4 + CCR6 + CCR4 + CXCR3 - Combined sorting of T4 Th17 cells;

[0010] Application of CD45 + CD3 + CD8 + CCR7 + CD45RA + Combined sorting of T8 naive cells;

[0011] Application of CD45 + CD3 + CD8 + CCR7 + CD45RA - Combined sorting of T8 TCM cells (Central Memory CD8 + TCells);

[0012] Application of CD45 + CD3 + CD8 + CCR7 - CD45RA - Combined sorting of T8 TEM cells (Effector Memory CD8 + TCells);

[0013] Application of CD45 + CD3 + CD8 +CCR7 - CD45RA + Combined sorting of T8 TEMRA cells;

[0014] Application of CD45 + CD3 + CD4 + CCR7 + CD45RA + Combined sorting T4 cell;

[0015] Application of CD45 + CD3 + CD4 + CCR7 + CD45RA - Combined sorting of T4 TCM cells (Central Memory CD4 + TCells);

[0016] Application of CD45 + CD3 + CD4 + CCR7 - CD45RA - Combined sorting of T4 TEM cells (Effector Memory CD4 + TCells);

[0017] Application of CD45 + CD3 + CD4 + CCR7 - CD45RA + Combined sorting of T4 TEMRA cells;

[0018] Application of CD45 + CD3 + TCR + Combined sorting of γδT cells;

[0019] Application of CD45 + CD3 - CD19 + CD27 - Combined sorting of B Naive cells;

[0020] Application of CD45 + CD3 - CD19 + CD27 + Combined sorting of B Memory cells;

[0021] Application of CD45 + CD3- CD19 - CD14 - CD56 + Combined sorting of Natural Killer Cells (NK);

[0022] Application of CD45 + CD3 - CD19 - CD14 + CD16 + Combined sorting of Monocyte N monocytes;

[0023] Application of CD45 + CD3 - CD19 - CD14 + CD16 - Combined sorting of Monocyte C monocytes;

[0024] Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD123 + Combined sorting of plasmacytoid dendritic cells (pDCs);

[0025] Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD11c + Combined sorting of myeloid dendritic cells (mDCs);

[0026] Application of CD45 int CD203c + Combined sorting of basophils (Basophil);

[0027] Application of CD45 int CD16 dim CD123 + Combined sorting of eosinophils;

[0028] Application of CD45 int CD16 + CD123 - CD62L +Combined sorting of neutrophils; or

[0029] Application of CD45 + CD11b + CD33 + HLADR - Combinatorial sorting of myeloid-derived suppressor cells (MDSCs).

[0030] The method of the present invention can be used to efficiently utilize blood samples. For example, only 4 mL of blood can be used to sort out up to 24 different immune cell subtypes for use in the monitoring or testing of immune cells in clinical peripheral blood and in the multi-omics analysis process of cells in the scientific research field.

[0031] Furthermore, the method comprises sorting at least ten of the above-mentioned immune cells according to markers.

[0032] Furthermore, the method comprises sorting at least fifteen of the above immune cells according to markers.

[0033] Furthermore, the method comprises sorting at least twenty of the above-mentioned immune cells according to markers.

[0034] Furthermore, the method comprises sorting the following immune cells according to markers:

[0035] Application of CD45 + CD3 + CD4 + CD25 + CD127 low Combined sorting of regulatory T cells (Treg);

[0036] Application of CD45 + CD3 + CD4 + CCR6 - CCR4 - CXCR3 + Combined sorting of T4 Th1 cells;

[0037] Application of CD45 + CD3 + CD4 + CCR6 - CCR4 + CXCR3 - Combined sorting of T4 Th2 cells;

[0038] Application of CD45 +CD3 + CD4 + CCR6 + CCR4 + CXCR3 - Combined sorting of T4 Th17 cells;

[0039] Application of CD45 + CD3 + CD8 + CCR7 + CD45RA + Combined sorting of T8 naive cells;

[0040] Application of CD45 + CD3 + CD8 + CCR7 + CD45RA - Combined sorting of T8 TCM cells (Central Memory CD8 + TCells);

[0041] Application of CD45 + CD3 + CD8 + CCR7 - CD45RA - Combined sorting of T8 TEM cells (Effector Memory CD8 + TCells);

[0042] Application of CD45 + CD3 + CD8 + CCR7 - CD45RA + Combined sorting of T8 TEMRA cells;

[0043] Application of CD45 + CD3 + CD4 + CCR7 + CD45RA + Combined sorting T4 cell;

[0044] Application of CD45 + CD3 + CD4 + CCR7 + CD45RA - Combined sorting of T4 TCM cells (Central Memory CD4 + TCells);

[0045] Application of CD45 + CD3 + CD4 + CCR7 - CD45RA - Combined sorting of T4 TEM cells (Effector Memory CD4 + TCells);

[0046] Application of CD45 + CD3 + CD4 + CCR7 - CD45RA + Combined sorting of T4 TEMRA cells;

[0047] Application of CD45 + CD3 + TCR + Combined sorting of γδT cells;

[0048] Application of CD45 + CD3 - CD19 + CD27 - Combined sorting of B Naive cells;

[0049] Application of CD45 + CD3 - CD19 + CD27 + Combined sorting of B Memory cells;

[0050] Application of CD45 + CD3 - CD19 - CD14 - CD56 + Combined sorting of Natural Killer Cells (NK);

[0051] Application of CD45 + CD3 - CD19 - CD14 + CD16 + Combined sorting of Monocyte N monocytes;

[0052] Application of CD45 + CD3 - CD19 - CD14 + CD16 - Combined sorting of Monocyte C monocytes;

[0053] Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD123 + Combined sorting of plasmacytoid dendritic cells (pDCs);

[0054] Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD11c + Combined sorting of myeloid dendritic cells (mDCs);

[0055] Application of CD45 int CD203c + Combined sorting of basophils (Basophil);

[0056] Application of CD45 int CD16 dim CD123 + Combined sorting of eosinophils;

[0057] Application of CD45 int CD16 + CD123 - CD62L + Combination sorting of neutrophils; and

[0058] Application of CD45 + CD11b + CD33 + HLADR - Combinatorial sorting of myeloid-derived suppressor cells (MDSCs).

[0059] By sorting with the above-mentioned markers, 24 immune cell types can be detected simultaneously based on a 15-color flow sorter, and the corresponding cell types can be directly sorted out.

[0060] Furthermore, the above immune cells were divided into four groups and tested simultaneously.

[0061] In some specific embodiments, the four groups are:

[0062] Group 1: Treg cells, T4 Th1 cells, T4 Th2 cells, T4 Th17 cells;

[0063] Group 2: T8 naive cells, T8 TCM cells, T8 TEM cells, T8 TEMRA cells, γδT cells, T4 T4 TCM cells, T4 TEM cells, T4 TEMRA cells;

[0064] Group III: B naive cells, B memory cells, natural killer cells, Monocyte N monocytes, Monocyte C monocytes, plasmacytoid dendritic cells, myeloid dendritic cells;

[0065] Group 4: basophils, eosinophils, neutrophils, myeloid-derived suppressor cells.

[0066] Furthermore, the above immune cell sorting method is for non-diagnostic purposes.

[0067] Furthermore, the immune cells are derived from blood, in particular, from peripheral blood.

[0068] Furthermore, the immune cell sorting comprises the following steps:

[0069] S1. Collect blood samples, in particular, peripheral blood samples;

[0070] S2. Mix the blood sample and PBS solution in equal volumes; then transfer the blood sample to the surface of lymphocyte separation solution and perform density gradient centrifugation;

[0071] Preferably, the volume of the lymphocyte separation fluid is 1.5 times the volume of the peripheral blood sample;

[0072] Preferably, the density gradient centrifugation is performed at 350 g for 30 minutes at room temperature.

[0073] S3, taking out the mononuclear cell layer cells obtained in step S2, washing them twice with PBS and counting the cells, centrifuging them, and removing the PBS supernatant;

[0074] S4, resuspending the mononuclear cell layer obtained in step S3 in flow cytometry staining solution, adding Fc blocking agent and mixing, and incubating in the dark for 10 minutes;

[0075] S5, dividing the above immune cells into four groups for flow cytometry antibody staining;

[0076] S6. Streamline the machine and analyze the results.

[0077] Furthermore, the mononuclear cell layer is peripheral blood mononuclear cells, PBMC.

[0078] The immune cells of the present invention are derived from the mononuclear cell layer.

[0079] Furthermore, the specific dyeing mode of step S5 is:

[0080] The first group was added with flow cytometry antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, and CXCR3-BV510;

[0081] The second group added flow cytometry antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, and TCRgd-BV421;

[0082] The third group added flow cytometry antibodies CD45-APC, CD3-APC / Cy7, CD19-BV510, CD27-BV421, CD14-BB700, CD16-BV605, HLA-DR-PE, CD56-PE / Cy7, CD123-PE / Cy5, and CD11c-FITC;

[0083] The fourth group added flow cytometry antibodies CD45-APC, CD16-BV605, HLA-DR-PE, CD123-PE / Cy5, CD33-BV510, CD11b-PE / Cy7, CD203c-BB700, and CD62L-BV421.

[0084] Furthermore, before adding the above-mentioned flow cytometry antibody, the second group also added CCR7-BV510 flow cytometry antibody.

[0085] CD45RO-PE / DZ594 flow cytometry antibody can assist in identifying four types of immune cells, namely, CD45 + CD3 + CD4 - CD8 + CCR7 + CD45RO - CD45RA + Combination of assisted identification of T8 naive cells; application of CD45 + CD3 + CD4 - CD8 + CCR7 + CD45RO + CD45RA- Combination-assisted identification of T8 TCM cells; application of CD45 + CD3 + CD4 - CD8 + CCR7 - CD45RO + CD45RA - Effector Memory CD8 + T Cells); application of CD45 + CD3 + CD4 - CD8 + CCR7 - CD45RO - CD45RA + Combination-assisted identification of T8 TEMRA cells.

[0086] Furthermore, between step S5 and step S6, there are the following steps:

[0087] 1) Removal of red blood cells from the cell sample; or

[0088] 2) Add dead cell dyes, such as 7-AAD, to screen dead cells.

[0089] In yet another aspect, the present invention provides use of the above-mentioned immune cell sorting method in preparing an immune cell sorting kit.

[0090] In another aspect, the present invention provides an immune cell sorting kit, comprising:

[0091] Group 1: antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, CXCR3-BV510;

[0092] Group 2: antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, TCRgd-BV421;

[0093] Group 3: antibodies CD45-APC, CD3-APC / Cy7, CD19-BV510, CD27-BV421, CD14-BB700, CD16-BV605, HLA-DR-PE, CD56-PE / Cy7, CD123-PE / Cy5, CD11c-FITC;

[0094] Group 4: Antibodies CD45-APC, CD16-BV605, HLA-DR-PE, CD123-PE / Cy5, CD33-BV510, CD11b-PE / Cy7, CD203c-BB700, CD62L-BV421. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 This is a flow cytometry plot of 8 T cell subsets in the peripheral blood of a patient with multiple myeloma;

[0096] Figure 2 Proteomic characterization results of eight T cell subtypes sorted from peripheral blood samples of five patients with multiple myeloma;

[0097] Figure 3 This is a flow cytometry plot of 24 immune cell subsets in the peripheral blood of leukemia patients;

[0098] Figure 4 This is a flow cytometry plot of 24 immune cell subsets in a healthy human leukocyte filter disc cell sample; DETAILED DESCRIPTION

[0099] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0100] In the following examples, the present invention uses lymphocyte separation solution (GE Healthcare), red blood cell lysis solution (BDPharm Lyse TM ), fluorescently labeled antibody reagents (both BioLegend and BD used) as separation reagents, and flow cytometer (BD FACSAria SORP) as separation instrument, sorting experiments were carried out on all or part of the cell types of the 24 immune cells.

[0101] Example 1: Isolation of 8 T cell subtypes from peripheral blood samples of patients with multiple myeloma

[0102] This embodiment provides a method for sorting T cell subtypes from peripheral blood samples of multiple myeloma patients. The sample used in this embodiment is about 4 mL of the remaining peripheral blood sample of multiple myeloma patients collected in clinical EDTA anticoagulation tubes. The specific operation steps are:

[0103] 1. Take the peripheral blood sample out of the anticoagulant tube and mix it evenly with an equal volume of 1× PBS solution; then transfer it to the surface of the lymphocyte separation solution and immediately perform density gradient centrifugation. The centrifugation conditions are: room temperature, speed 350g, 30 minutes, and the acceleration and deceleration gears of the centrifuge are both set to "3".

[0104] 2. After centrifugation, the sample can be seen to be stratified. Remove the buffy coat layer, i.e. PBMC, wash it twice with PBS and count the cells. Centrifuge at 300g for 5 minutes and remove the PBS supernatant.

[0105] 3. Resuspend PBMC in flow cytometry staining solution, add Fc blocking agent, mix well, and incubate in the dark for 10 minutes.

[0106] 4. Divide the above PBMCs into two equal parts for flow antibody staining, and control the volume of each part at about 100 μL. Add flow antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, and CXCR3-BV510 to the first part; add flow antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, and CD45RO-PE / DZ594 to the second part. Incubate at room temperature away from light for 15-20 minutes.

[0107] 5. If there are red blood cells mixed in the PBMC, add red blood cell lysis buffer, incubate for several minutes in the dark, add 2 times the volume of PBS solution to terminate the reaction, centrifuge at 300g for 5 minutes, and discard the supernatant.

[0108] 6. Use more than 2 mL of staining solution to wash away excess antibodies in the system.

[0109] 7. Take 10 7 Resuspend PBMCs at a concentration of 100 μL / mL, with a total volume of no less than 500 μL. Add 5 μL 7-AAD and sort them on the machine after 5-10 minutes.

[0110] The results are shown as Figure 1 ,Treg, Th1, Th2, Th17, T8 Eight T cell subsets, including T8 CM, T8 EM, and T8EMRA, were sorted out simultaneously in one experiment, indicating that eight types of T cells can be accurately and efficiently sorted out from limited clinical residual samples. This embodiment sorted out a total of five clinical residual samples, and the number of cells and the corresponding protein amount of each cell type were counted. The eight cell subsets sorted out in this embodiment were subsequently used as proteomics experimental materials, and 4030, 3979, 3968, and 3957 proteins were identified from the four T cell subtypes of Treg, Th1, Th2, and Th17, respectively. 4063, 3843, 4138, and 4078 proteins were identified in the four T cell subtypes, T8 CM, T8 EM, and T8 EMRA, respectively. Figure 2 ).

[0111] Example 2: Isolation of 24 Immune Cell Subtypes from Peripheral Blood Samples of Leukemia Patients

[0112] This embodiment provides a method for sorting immune cell subtypes from peripheral blood samples of leukemia patients. The samples used in this embodiment are residual peripheral blood samples of leukemia patients collected in clinical heparin anticoagulation tubes.

[0113] The specific steps are:

[0114] 1. Take the peripheral blood sample out of the anticoagulant tube and mix it evenly with an equal volume of PBS solution; then transfer it to the surface of the lymphocyte separation solution and immediately perform density gradient centrifugation. The centrifugation conditions are: room temperature, speed 350g, 30 minutes, and the acceleration and deceleration gears of the centrifuge are both set to "3".

[0115] 2. After centrifugation, the sample can be seen to be stratified. Take out the buffy coat layer, i.e. PBMC. Combine the buffy coat layers of the two samples, wash twice with PBS and count the cells. Centrifuge at 300g for 5 minutes and remove the PBS supernatant.

[0116] 3. Resuspend PBMC in flow cytometry staining solution, add Fc blocking agent, mix well, and incubate in the dark for 10 minutes.

[0117] 4. The above PBMCs were divided into four equal parts for flow cytometry antibody staining. The volume of each part was controlled at about 100 μL. The first part was added with flow cytometry antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, and CXCR3-BV510; the second part was added with flow cytometry antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, TCRgd-BV421, and CD45RO-PE / DZ594 ; The third portion was added with flow cytometry antibodies CD45-APC, CD3-APC / Cy7, CD19-BV510, CD27-BV421, CD14-BB700, CD16-BV605, HLA-DR-PE, CD56-PE / Cy7, CD123-PE / Cy5, and CD11c-FITC; the fourth portion was added with flow cytometry antibodies CD45-APC, CD16-BV605, HLA-DR-PE, CD123-PE / Cy5, CD33-BV510, CD11b-PE / Cy7, CD203c-BB700, and CD62L-BV421 and incubated at room temperature away from light for 15-20 minutes.

[0118] 5. If there are red blood cells mixed in the PBMC, add red blood cell lysis buffer, incubate for several minutes in the dark, add 2 times the volume of PBS solution to terminate the reaction, centrifuge at 300g for 5 minutes, and discard the supernatant.

[0119] 6. Use more than 2 mL of staining solution to wash away excess antibodies in the system.

[0120] 7. Take 10 7 Resuspend PBMCs at a concentration of 100 μL / mL, with a total volume of no less than 500 μL. Add 5 μL 7-AAD and sort them on the machine after 5-10 minutes.

[0121] The results are shown as Figure 3 ,Treg, Th1, Th2, Th17, T8 T8 CM, T8 EM, T8EMRA, γδT cells, T4 T4 TCM, T4 TEM, T4 TEMRA, B naive, B memory, NK, Monocyte N, Monocyte C, Dendritic Cells Plasmacytoid, Dendritic Cells Myeloid, Basophil, Eosinophils, Neutrophils, MDSC and other 24 immune cell subsets were sorted out simultaneously in one experiment, indicating that 24 immune cells can be accurately and efficiently sorted out from limited clinical residual samples. In this embodiment, 2 clinical residual samples were combined and sorted, and the 24 cell subsets sorted out in this embodiment can also be used as proteomics experimental materials.

[0122] Example 3: Sorting of 24 immune cell subtypes in healthy human leukocyte filter plate cell samples

[0123] This embodiment provides a method for sorting immune cell subtypes from a healthy human leukocyte filter disc cell sample. The sample used in this embodiment is a healthy human leukocyte filter disc sample. The specific operation steps are:

[0124] 1. Identify the flow direction of the liquid in the cell filter, observe the direction of the arrow on the plastic protrusion on the outside of the filter, hold the outflow port marked with the arrow on the filter, use medical scissors to cut off the inflow port of the filter first, and then cut off the outflow port. Use a syringe to extract 50mL of PBS solution and slowly wash the cells in the plate into the centrifugal collection tube. Centrifuge the washed blood cells at 18℃, 2000g, 10 minutes, and set the acceleration gear of the centrifuge to "9" and the deceleration gear to "5". After centrifugation, discard the supernatant and resuspend the cell pellet with PBS solution.

[0125] 2. Slowly transfer the blood cell sample resuspended in step 1 to the surface of the lymphocyte separation solution, centrifuge at 18°C, 500g, for 30 minutes, with the acceleration gear of the centrifuge set to "4" and the deceleration gear set to "0". Collect the PBMC layer.

[0126] 3. Wash the PBMC cells collected in step 2 with PBS solution, centrifuge at 500g for 10 minutes at room temperature, aspirate the supernatant, resuspend the cells in PBS again, centrifuge at 160g for 10 minutes at room temperature, and repeat this step once. Resuspend the cells with 10mL of room temperature PBS, place on ice, and count the cells. This step is to remove platelets.

[0127] 4. Freeze the PBMC samples. Prepare the cell freezing solution with 90% FBS + 10% DMSO. Centrifuge the PBMC samples washed in step 3 at 500g for 10 minutes at 4°C. Resuspend the cells in the pre-cooled cell freezing solution to a concentration of 5×10 6 / mL, dispense into cell freezing tubes, gradually cool down to -80℃ refrigerator and store until thawed when needed.

[0128] 5. Resuscitate PBMC samples. Prepare complete culture medium with RPMI-1640 + 10% FBS + 1% Penicillin / Streptomycin. Thaw the frozen PBMC samples in step 4 in a 37°C water bath. Slowly resuspend the cells in complete culture medium, centrifuge at 500g for 10 minutes at room temperature, and remove the supernatant. Wash the cell samples twice with PBS solution.

[0129] 6. Resuspend the PBMC sample revived in step 5 in flow cytometry staining solution, add Fc blocking agent, mix well, and incubate in the dark for 10 minutes.

[0130] 7. Divide the PBMC in step 6 into four parts for flow cytometry antibody staining. The volume of each part is controlled at about 100 μL. The first part is added with flow cytometry antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, CXCR3-BV510; the second part is added with flow cytometry antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, TCRgd-BV421, CD45RO-PE / DZ594 ; The third portion was added with flow cytometry antibodies CD45-APC, CD3-APC / Cy7, CD19-BV510, CD27-BV421, CD14-BB700, CD16-BV605, HLA-DR-PE, CD56-PE / Cy7, CD123-PE / Cy5, and CD11c-FITC; the fourth portion was added with flow cytometry antibodies CD45-APC, CD16-BV605, HLA-DR-PE, CD123-PE / Cy5, CD33-BV510, CD11b-PE / Cy7, CD203c-BB700, and CD62L-BV421 and incubated at room temperature away from light for 15-20 minutes.

[0131] 8. If there are red blood cells mixed in the PBMC, add red blood cell lysis buffer, incubate for a few minutes in the dark, add 2 times the volume of PBS solution to terminate the reaction, centrifuge at 300g for 5 minutes, and discard the supernatant.

[0132] 9. Use more than 2 mL of staining solution to wash away excess antibodies in the system.

[0133] 10. Take 10 7 Resuspend PBMCs at a concentration of 100 μL / mL, with a total volume of no less than 500 μL. Add 5 μL 7-AAD and sort them on the machine after 5-10 minutes.

[0134] The results are shown as Figure 4 ,Treg, Th1, Th2, Th17, T8 T8 CM, T8 EM, T8EMRA, γδT cells, T4 T4 TCM, T4 TEM, T4 TEMRA, B naive, B memory, NK, Monocyte N, Monocyte C, Dendritic Cells Plasmacytoid, Dendritic Cells Myeloid, Basophil, Eosinophils, Neutrophils, MDSC and other 24 immune cell subsets were sorted out simultaneously in one experiment, indicating that 24 immune cells can be accurately and efficiently sorted out from the sample preserved in the leukocyte filter disk. The 24 cell subsets sorted out in this embodiment can be used as proteomics experimental materials.

[0135] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for sorting immune cells, comprising sorting at least eight of the following immune cells according to markers: Application of CD45 + CD3 + CD4 + CD25 + CD127 low Combinatorial sorting of regulatory T cells; Application of CD45 + CD3 + CD4 + CCR6 - CCR4 - CXCR3 + Combined sorting of T4 Th1 cells; Application of CD45 + CD3 + CD4 + CCR6 - CCR4 + CXCR3 - Combined sorting of T4 Th2 cells; Application of CD45 + CD3 + CD4 + CCR6 + CCR4 + CXCR3 - Combined sorting of T4 Th17 cells; Application of CD45 + CD3 + CD8 + CCR7 + CD45RA + Combined sorting of T8 naive cells; Application of CD45 + CD3 + CD8 + CCR7 + CD45RA - Combined sorting of T8 TCM cells; Application of CD45 + CD3 + CD8 + CCR7 - CD45RA - Combined sorting of T8 TEM cells; Application of CD45 + CD3 + CD8 + CCR7 - CD45RA + Combined sorting of T8 TEMRA cells; Application of CD45 + CD3 + CD4 + CCR7 + CD45RA + Combined sorting T4 cell; Application of CD45 + CD3 + CD4 + CCR7 + CD45RA - Combined sorting of T4 TCM cells; Application of CD45 + CD3 + CD4 + CCR7 - CD45RA - Combined sorting of T4 TEM cells; Application of CD45 + CD3 + CD4 + CCR7 - CD45RA + Combined sorting of T4 TEMRA cells; Application of CD45 + CD3 + TCR + Combined sorting of γδT cells; Application of CD45 + CD3 - CD19 + CD27 - Combined sorting of B naive cells; Application of CD45 + CD3 - CD19 + CD27 + Combined sorting of B memory cells; Application of CD45 + CD3 - CD19 - CD14 - CD56 + Combinatorial sorting of natural killer cells; Application of CD45 + CD3 - CD19 - CD14 + CD16 + Combined sorting of Monocyte N monocytes; Application of CD45 + CD3 - CD19 - CD14 + CD16 - Combined sorting of Monocyte C monocytes; Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD123 + Combinatorial sorting of plasmacytoid dendritic cells; Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD11c + Combination sorting of cells myeloid dendritic cells; Application of CD45 int CD203c + Combined sorting of basophils; Application of CD45 int CD16 dim CD123 + Combined sorting of eosinophils; Application of CD45 int CD16 + CD123 - CD62L + Combined sorting of neutrophils; or Application of CD45 + CD11b + CD33 + HLADR - Combinatorial sorting of myeloid-derived suppressor cells.

2. The immune cell sorting method according to claim 1, characterized in that: At least ten, fifteen, or twenty of the immune cells are sorted.

3. The immune cell sorting method according to claim 2, characterized in that: This includes sorting the following immune cells by markers: Application of CD45 + CD3 + CD4 + CD25 + CD127 low Combinatorial sorting of regulatory T cells; Application of CD45 + CD3 + CD4 + CCR6 - CCR4 - CXCR3 + Combined sorting of T4 Th1 cells; Application of CD45 + CD3 + CD4 + CCR6 - CCR4 + CXCR3 - Combined sorting of T4 Th2 cells; Application of CD45 + CD3 + CD4 + CCR6 + CCR4 + CXCR3 - Combined sorting of T4 Th17 cells; Application of CD45 + CD3 + CD8 + CCR7 + CD45RA + Combined sorting of T8 naive cells; Application of CD45 + CD3 + CD8 + CCR7 + CD45RA - Combined sorting of T8 TCM cells; Application of CD45 + CD3 + CD8 + CCR7 - CD45RA - Combined sorting of T8 TEM cells; Application of CD45 + CD3 + CD8 + CCR7 - CD45RA + Combined sorting of T8 TEMRA cells; Application of CD45 + CD3 + CD4 + CCR7 + CD45RA + Combined sorting T4 cell; Application of CD45 + CD3 + CD4 + CCR7 + CD45RA - Combined sorting of T4 TCM cells; Application of CD45 + CD3 + CD4 + CCR7 - CD45RA - Combined sorting of T4 TEM cells; Application of CD45 + CD3 + CD4 + CCR7 - CD45RA + Combined sorting of T4 TEMRA cells; Application of CD45 + CD3 + TCR + Combined sorting of γδT cells; Application of CD45 + CD3 - CD19 + CD27 - Combined sorting of B naive cells; Application of CD45 + CD3 - CD19 + CD27 + Combined sorting of B memory cells; Application of CD45 + CD3 - CD19 - CD14 - CD56 + Combinatorial sorting of natural killer cells; Application of CD45 + CD3 - CD19 - CD14 + CD16 + Combined sorting of Monocyte N monocytes; Application of CD45 + CD3 - CD19 - CD14 + CD16 - Combined sorting of Monocyte C monocytes; Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD123 + Combinatorial sorting of plasmacytoid dendritic cells; Application of CD45 + CD3 - CD19 - CD14 - HLADR + CD11c + Combination sorting of cells myeloid dendritic cells; Application of CD45 int CD203c + Combined sorting of basophils; Application of CD45 int CD16 dim CD123 + Combined sorting of eosinophils; Application of CD45 int CD16 + CD123 - CD62L + Combined sorting of neutrophils; as well as Application of CD45 + CD11b + CD33 + HLADR - Combinatorial sorting of myeloid-derived suppressor cells.

4. The immune cell sorting method according to claim 3, characterized in that: Immune cells were divided into four groups, which are: Group 1: Treg cells, T4 Th1 cells, T4 Th2 cells, T4 Th17 cells; Group 2: T8 naive cells, T8 TCM cells, T8 TEM cells, T8 TEMRA cells, γδT cells, T4 T4 TCM cells, T4 TEM cells, T4 TEMRA cells; Group III: B naive cells, B memory cells, natural killer cells, Monocyte N monocytes, Monocyte C monocytes, plasmacytoid dendritic cells, myeloid dendritic cells; Group 4: basophils, eosinophils, neutrophils, myeloid-derived suppressor cells.

5. The immune cell sorting method according to any one of claims 1 to 4, characterized in that: The immune cell sorting method further comprises the following steps: S1. Collect blood samples; S2. Mix the blood sample with an equal volume of PBS solution; transfer it to the surface of lymphocyte separation solution and perform density gradient centrifugation; S3, taking out the mononuclear cell layer cells obtained in step S2, washing with PBS and counting the cells, centrifuging, and removing the PBS supernatant; S4, resuspend the immune cells obtained in step S3, add Fc blocking agent and mix well, and incubate in dark for 10 minutes; S5, dividing the above immune cells into four groups for antibody staining; S6. Analysis results.

6. The immune cell sorting method according to claim 5, characterized in that: The dyeing mode of step S5 is: In the first group, antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, and CXCR3-BV510 were added; The second group was added with antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, and TCRgd-BV421; The third group was added with antibodies CD45-APC, CD3-APC / Cy7, CD19-BV510, CD27-BV421, CD14-BB700, CD16-BV605, HLA-DR-PE, CD56-PE / Cy7, CD123-PE / Cy5, and CD11c-FITC; The fourth group added antibodies CD45-APC, CD16-BV605, HLA-DR-PE, CD123-PE / Cy5, CD33-BV510, CD11b-PE / Cy7, CD203c-BB700, and CD62L-BV421.

7. The immune cell sorting method according to claim 6, characterized in that: The second group was given CCR7-BV510 antibody before the above antibodies.

8. The immune cell sorting method according to claim 7, characterized in that: Between step S5 and step S6, there are the following steps: 1) Removing red blood cells from the cell sample; and / or 2) Add dead cell dye to screen dead cells.

9. Use of the immune cell sorting method according to any one of claims 1 to 8 in preparing an immune cell sorting kit.

10. An immune cell sorting kit, comprising: Group 1: antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD25-BV421, CD127-PE / DZ594, CCR6-APC, CCR4-PE / Cy7, CXCR3-BV510; Group 2: antibodies CD45-APC / Cy7, CD3-FITC, CD4-PE, CD8-PE / Cy7, CCR7-BV510, CD45RA-APC, TCRgd-BV421; Group 3: antibodies CD45-APC, CD3-APC / Cy7, CD19-BV510, CD27-BV421, CD14-BB700, CD16-BV605, HLA-DR-PE, CD56-PE / Cy7, CD123-PE / Cy5, CD11c-FITC; Group 4: Antibodies CD45-APC, CD16-BV605, HLA-DR-PE, CD123-PE / Cy5, CD33-BV510, CD11b-PE / Cy7, CD203c-BB700, CD62L-BV421.

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