CN113866409A - Kit for simultaneously detecting various cell subsets and functional changes and application thereof - Google Patents

Kit for simultaneously detecting various cell subsets and functional changes and application thereof Download PDF

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CN113866409A
CN113866409A CN202111164758.5A CN202111164758A CN113866409A CN 113866409 A CN113866409 A CN 113866409A CN 202111164758 A CN202111164758 A CN 202111164758A CN 113866409 A CN113866409 A CN 113866409A
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cell
cells
parts
kit
antibody composition
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戚何妹
陶铸
容志恩
亢中奎
丁文婷
秦莉
陈小平
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Zhongke Lanhua Guangzhou Biomedical Technology Co ltd
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Guangzhou Cas Lamvac Biotech Co Ltd
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2015/1006—Investigating individual particles for cytology
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    • G01—MEASURING; TESTING
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    • G01N2469/10—Detection of antigens from microorganism in sample from host

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Abstract

The invention provides a kit for simultaneously detecting various cell subsets and functional changes and application thereof, wherein the kit comprises an antibody composition, and the antibody composition comprises a surface antibody composition and an intracellular antibody composition; the surface antibody composition comprises a combination of at least two of Zombie NIR, CD45, Ly6G, CD11b, F4/80, Ly6c, NK1.1, CD3, CD4, CD25, CD8a, CD19, CD11c, CD80, or CD 206; the intrabody composition includes a combination of at least two of FoxP3, IL-10, Granzyme B, Perforin, or IFN- γ. The invention also provides a method for detecting the change of the immune cell subpopulation. The kit can be used for simultaneously detecting the change of a plurality of immune cell subsets and functions, and the result is comprehensive and specific.

Description

Kit for simultaneously detecting various cell subsets and functional changes and application thereof
Technical Field
The invention belongs to the technical field of immune level detection, and particularly relates to a kit for simultaneously detecting various cell subsets and functional changes and application thereof.
Background
Flow cytometry is a technique for the continuous detection of individual cells or cell-size-like particles in a population of cells that allows for the rapid, high-throughput, accurate, simultaneous detection of multiple parameters of the cells or particles. At present, the flow technology is widely applied to scientific research such as hematology, immunology, oncology and the like or clinical related inspection fields, and is one of important detection methods.
The disease is often accompanied by changes in the immune level of the body during the development process, such as the significant changes in the main immune cell subsets in blood, spleen and tumor tissues during the development process of tumors, including CD 4T cells, CD 8T cells, Treg cells, granulocytes, monocytes, macrophages, B cells, NK cells, MDSC cells, DC cells and the like. The change difference of various immune cells is detected, the difference of an immune map is drawn, the method has important effects on revealing the pathogenesis of diseases including tumors, the relation between the diseases and the change of the immune level of an organism, the effectiveness of disease treatment measures and the like, can be used for judging the disease process and the treatment effect, and is expected to screen a proper prognostic index. Therefore, a set of streaming detection schemes covering the whole mark has important significance.
In the existing flow detection technology, the simultaneous detection of multiple cell subsets in one detection is difficult to realize. Therefore, it is a problem to be solved how to provide a product and a method capable of simultaneously detecting multiple cell subsets.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides the kit for simultaneously detecting multiple cell subsets and function changes and the application thereof, the change conditions of multiple immune cell subsets can be confirmed in one detection reaction through the mutual matching of different antibodies in the kit, the function changes of immune cells in an organism can be comprehensively analyzed, the analysis is more comprehensive, and the detection efficiency is higher.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a kit for simultaneously detecting multiple cell subsets and functional changes, the kit for simultaneously detecting multiple cell subsets and functional changes comprising an antibody composition;
the antibody compositions include surface antibody compositions and intrabody compositions;
the surface antibody composition comprises a combination of at least two of Zombie NIR, CD45, Ly6G, CD11b, F4/80, Ly6c, NK1.1, CD3, CD4, CD25, CD8a, CD19, CD11c, CD80, or CD 206;
the intrabody composition includes a combination of at least two of FoxP3, IL-10, Granzyme B, Perforin, or IFN- γ.
In the invention, the kit contains the surface antibody composition and the intracellular antibody composition, and can simultaneously mark a plurality of antibodies in flow detection, namely, the functional changes of a plurality of immune cells can be simultaneously detected in one detection reaction, so that the comprehensive and specific analysis of the change difference of main immune cells in the disease occurrence process is facilitated, the analysis result is more comprehensive and accurate, the detection efficiency is higher, and the kit has important significance for the diagnosis of the disease and the monitoring of the disease process.
In the present invention, the surface antibody composition detects receptors on the surface of a cell body, and the intrabody composition detects intracellular cytokines.
According to the kit for simultaneously detecting multiple cell subsets and functional changes, the use is convenient, the operation is simple, and the detection result is comprehensive and accurate.
Preferably, the proportion by weight of the Zombie NIR in the antibody composition is 0.05-0.2 parts, for example, 0.05 part, 0.1 part, 0.15 part or 0.2 part, and other specific values within the range of values can be selected, and are not described herein again.
Preferably, the weight part of the CD45 in the antibody composition is 0.2 to 0.5 part, for example, 0.2 part, 0.25 part, 0.3 part, 0.35 part, 0.4 part, 0.45 part, or 0.5 part, and other specific values in the value range can be selected, and are not repeated herein.
Preferably, the part by weight of the Ly6G in the antibody composition is 0.2 to 0.5 part, for example, 0.2 part, 0.25 part, 0.3 part, 0.35 part, 0.4 part, 0.45 part, or 0.5 part, and the like, and other specific values within the range of values can be selected, and are not repeated herein.
Preferably, the weight part of the CD11b in the antibody composition is 0.2 to 0.5 part, for example, 0.2 part, 0.25 part, 0.3 part, 0.35 part, 0.4 part, 0.45 part, or 0.5 part, and other specific values in the value range can be selected, and are not repeated herein.
Preferably, the weight part of the F4/80 in the antibody composition is 0.5 to 0.7 part, for example, 0.5 part, 0.55 part, 0.6 part, 0.65 part, or 0.7 part, and other specific values in the value range can be selected, and are not repeated herein.
Preferably, the part by weight of the Ly6c in the antibody composition is 0.05-0.1 part, for example, 0.05 part, 0.06 part, 0.07 part, 0.08 part, 0.09 part, or 0.1 part, and other specific values in the value range can be selected, and are not repeated herein.
Preferably, the weight part of the NK1.1 in the antibody composition is 0.1 to 0.8, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 or 0.8, and other specific values in the range of values can be selected, and are not repeated herein.
Preferably, the weight part of the CD3 in the antibody composition is 0.1 to 0.5 part, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part or 0.5 part, and other specific values within the value range can be selected, which is not described herein again.
Preferably, the weight part of the CD4 in the antibody composition is 0.1 to 0.3 part, for example, 0.1 part, 0.15 part, 0.2 part, 0.25 part or 0.3 part, and other specific values within the value range can be selected, which is not described herein again.
Preferably, the weight part of the CD25 in the antibody composition is 0.05 to 0.3 part, for example, 0.05 part, 0.1 part, 0.15 part, 0.2 part, 0.25 part, or 0.3 part, and other specific values in the value range can be selected, and are not repeated herein. Preferably, the weight part of the CD8a in the antibody composition is 0.1 to 0.5, for example, 0.1, 0.2, 0.3, 0.4, or 0.5, and other specific values in the value range can be selected, and are not repeated herein.
Preferably, the weight part of the CD19 in the antibody composition is 0.1 to 0.5 part, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, or 0.5 part, and other specific values within the value range can be selected, and are not repeated herein.
Preferably, the weight part of the CD11c in the antibody composition is 0.5 to 3 parts, for example, 0.5 part, 1 part, 1.5 parts, 2 parts, 2.5 parts, or 3 parts, and other specific values within the value range can be selected, and are not repeated herein.
Preferably, the weight part of the CD80 in the antibody composition is 1 to 4 parts, for example, 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, 3.5 parts, or 4 parts, and other specific values within the value range can be selected, and are not repeated herein.
Preferably, the weight part of the CD206 in the antibody composition is 0.5 to 3 parts, for example, 0.5 part, 1 part, 1.5 part, 2 parts, 2.5 parts, or 3 parts, and other specific values within the value range can be selected, and are not repeated herein.
Preferably, the weight part of the FoxP3 in the antibody composition is 0.3 to 0.8, for example, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, and other specific values within the range can be selected, and are not repeated herein.
Preferably, the weight part of the IL-10 in the antibody composition is 2 to 5 parts, for example, 2 parts, 2.5 parts, 3 parts, 3.5 parts, 4 parts, 4.5 parts, or 5 parts, and other specific values within the value range can be selected, and are not repeated herein.
Preferably, the part by weight of the Granzyme B in the antibody composition is 0.1 to 0.5 part, for example, 0.1 part, 0.2 part, 0.3 part, 0.4 part, or 0.5 part, and other specific values within the numerical range can be selected, and are not repeated herein.
Preferably, the parts by weight of the Perforin in the antibody composition is 3 to 8 parts, for example, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, 5.5 parts, 6 parts, 6.5 parts, 7 parts, 7.5 parts, or 8 parts, and other specific values in the numerical range can be selected, and are not repeated herein.
Preferably, the parts by weight of the IFN- γ in the antibody composition are 3 to 8 parts, for example, 3 parts, 3.5 parts, 4 parts, 4.5 parts, 5 parts, 5.5 parts, 6 parts, 6.5 parts, 7 parts, 7.5 parts, or 8 parts, and other specific values in the numerical range can be selected, and are not repeated herein.
Preferably, the antibody composition comprises, in parts by weight: 0.05-0.2 part of Zombie NIR, 450.2-0.5 part of CD, 0.2-0.5 part of Ly6G 0.2, 0.2-0.5 part of CD11B 0.2, 0.2-0.5 part of F4/800.5-0.7 part of Ly6c 0.05, 0.05-0.1 part of NK1.10.1-0.8 part of CD 30.1-0.5 part of CD 40.1-0.3 part of CD 250.05-0.3 part of CD8a 0.1, 0.1-0.5 part of Granzyme B, 0.1-0.5 part of Perform 3-8 parts of IFN-gamma 3-8 parts of CD 190.1-0.5 part of CD11c 0.5, 0.5-3 parts of CD 801-4 parts of CD 2060.5-3 parts of FoxPb 30.3-0.8 part of IL-102-5 parts of Perform B, 0.1-0.5 part of Perform B, 3-8 parts of IFN-gamma 3 parts of.
As a preferred technical scheme, the kit for simultaneously detecting multiple cell subsets and functional changes comprises the following components in parts by weight: 0.08-0.15 part of Zombie NIR, 450.2-0.4 part of CD, 0.2-0.4 part of Ly6G 0.2, 0.2-0.4 part of CD11B 0.2, 0.2-0.4 part of F4/800.5-0.65 part of Ly6c 0.06, 0.06-0.1 part of NK1.10.4-0.7 part of CD 30.1-0.4 part of CD 40.1-0.2 part of CD 250.05-0.2 part of CD8a 0.1, 0.1-0.4 part of CD 190.1-0.4 part of CD11c 0.5, 0.5-2 part of CD 801, 3-2 parts of CD XP 2060.5, 0.3-0.6 part of Fonze B, 3-6 parts of IL-102-4 parts of Granze B, 3-6 parts of Perforin and 3-6 parts of IFN-gamma.
Preferably, the kit for simultaneously detecting multiple cell subsets and functional changes further comprises a blocking agent, a membrane-breaking solution and a machine-loading buffer solution.
In the invention, the upper machine buffer solution is flow cytometry solution. Preferably, the cells comprise any one of or a combination of at least two of CD 4T cells, CD 8T cells, Treg cells, Double negative T cells (Double negative T cells), granulocytes, macrophages, B cells, NK cells, MDSC cells or DC cells.
In a second aspect, the present invention provides a method for detecting a change in a subpopulation of immune cells, the method comprising detecting the change in the subpopulation of immune cells using the kit for simultaneously detecting a plurality of subpopulations of cells and a change in function according to the first aspect.
Preferably, the method comprises:
preparing single cell suspension, sealing with sealant, staining with surface antibody composition, washing, re-suspending cells, treating with membrane-breaking solution, adding intracellular antibody composition, incubating, removing supernatant, re-suspending with on-machine buffer solution, detecting on-machine, and analyzing.
According to the method, the change difference of various immune cells can be analyzed simultaneously in one detection reaction without additional operation steps, the efficiency is higher, the analysis result is more comprehensive, and the method has practical application value.
Preferably, the flow cytometer used in the on-machine detection is a full spectrum flow cytometer.
Preferably, the information of the full-spectrum flow cytometer is Cytek Aurora, 3-polar 28 channel.
Preferably, the analysis comprises:
removing cell debris from the cell compartment and then adherent cells;
setting multiple cell gates, and detecting the expression of multiple cell subsets.
Preferably, the analysis comprises in particular:
1. removing cell debris from the cell compartment and then adherent cells;
2. setting a CD45+ cell gate, and setting a plurality of gates to detect the expression condition of a plurality of groups of cells:
3. setting a Ly6G/SSC cell gate, and dividing the cell gate into a Ly6G + cell subgroup and a Ly 6G-cell subgroup;
(1) within the Ly6G + cell subset, the Ly6c/CD11b phylum was set, where CD11b + is neutrophils and Ly6c divides neutrophils into two subsets, Ly6c + and Ly6c-, the Ly6c + subset being the PMN-MDSC cell population;
(2) setting a F4/80/CD11b cytogate in Ly 6G-cytosubgroup, and dividing the cytoblast into four cytoblast subgroups of F4/80+ CD11b +, F4/80+ CD11b-, F4/80-CD11b + and F4/80-CD11 b-;
setting a CD80/CD206 cell gate within the F4/80+ CD11b + cell subset, wherein CD80+ CD 206-is macrophage subset M1 and CD80-CD206+ is macrophage subset M2;
(3) setting a F4/80/NK1.1 cytogate in Ly 6G-cell subgroup, and dividing the cells into four cell subgroups of F4/80+ NK1.1+, F4/80+ NK1.1-, F4/80-NK1.1+ and F4/80-NK 1.1-;
1) setting a CD3/SSC cell gate in a F4/80-NK1.1+ cell subset which is NK + cell subset, and dividing the cell gate into a CD3+ NK T cell subset and an NK cell subset;
2) within the F4/80-NK 1.1-cell subset, a CD3/SSC cell gate was set, divided into a CD3+ cell subset and a CD 3-cell subset;
setting a CD4/CD8 cytogate in a CD3+ T cell subgroup, and dividing the cell subgroup into three cell subgroups of CD8+ T cells, CD4+ T cells and DNT cells;
analyzing the expression of IFN-gamma, Perforin and Granzyme B in a CD8+ T cell subset;
setting a CD25/FoxP3 cytogate in a CD4+ T cell subset, wherein CD25+ FoxP3+ is a Tregs cell subset, and further analyzing the expression condition of IL-10 in the Tregs cell subset;
② setting a Ly6c/CD19 cytogate in the CD 3-cell subgroup, wherein CD19+ Ly6 c-is a B cell subgroup;
setting a CD11cCD11b cell gate within a CD19-Ly6c + cell subset, wherein CD11c + CD11b + is a Ly6c + DC cell subset and CD11c-CD11b + is an M-MDSC cell subset;
within the CD19-Ly6 c-cell subset, a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is a myeloid DC cell subset and CD11c + CD11b + is a plasma DC cell subset.
In the present invention, the analysis step can be specifically selected and analyzed according to the type of the sample to be detected.
As a preferred technical scheme, the method for detecting the change of the immune cell subpopulation comprises the following steps:
preparing single cell suspension, sealing with a sealing agent, staining with a surface antibody composition, washing, resuspending cells, adding a membrane breaking solution for treatment, adding an intracellular antibody composition, incubating, removing supernatant, resuspending with an on-machine buffer solution, performing on-machine detection with a full-spectrum flow cytometer, and analyzing;
the analysis comprises the following steps:
removing cell debris from the cell compartment and then adherent cells;
setting a plurality of cell gates, and detecting the expression conditions of a plurality of cell subsets;
the analysis specifically comprises:
1. removing cell debris from the cell compartment and then adherent cells;
2. setting a CD45+ cell gate, and setting a plurality of gates to detect the expression condition of a plurality of groups of cells:
3. setting a Ly6G/SSC cell gate, and dividing the cell gate into a Ly6G + cell subgroup and a Ly 6G-cell subgroup;
(1) within the Ly6G + cell subset, the Ly6c/CD11b phylum was set, where CD11b + is neutrophils and Ly6c divides neutrophils into two subsets, Ly6c + and Ly6c-, the Ly6c + subset being the PMN-MDSC cell population;
(2) setting a F4/80/CD11b cytogate in Ly 6G-cytosubgroup, and dividing the cytoblast into four cytoblast subgroups of F4/80+ CD11b +, F4/80+ CD11b-, F4/80-CD11b + and F4/80-CD11 b-;
setting a CD80/CD206 cell gate within the F4/80+ CD11b + cell subset, wherein CD80+ CD 206-is macrophage subset M1 and CD80-CD206+ is macrophage subset M2;
(3) setting a F4/80/NK1.1 cytogate in Ly 6G-cell subgroup, and dividing the cells into four cell subgroups of F4/80+ NK1.1+, F4/80+ NK1.1-, F4/80-NK1.1+ and F4/80-NK 1.1-;
1) setting a CD3/SSC cell gate in a F4/80-NK1.1+ cell subset which is NK + cell subset, and dividing the cell gate into a CD3+ NK T cell subset and an NK cell subset;
2) within the F4/80-NK 1.1-cell subset, a CD3/SSC cell gate was set, divided into a CD3+ cell subset and a CD 3-cell subset;
setting a CD4/CD8 cytogate in a CD3+ T cell subgroup, and dividing the cell subgroup into three cell subgroups of CD8+ T cells, CD4+ T cells and DNT cells;
analyzing the expression of IFN-gamma, Perforin and Granzyme B in a CD8+ T cell subset;
setting a CD25/FoxP3 cytogate in a CD4+ T cell subset, wherein CD25+ FoxP3+ is a Tregs cell subset, and further analyzing the expression condition of IL-10 in the Tregs cell subset;
② setting a Ly6c/CD19 cytogate in the CD 3-cell subgroup, wherein CD19+ Ly6 c-is a B cell subgroup;
setting a CD11cCD11b cell gate within a CD19-Ly6c + cell subset, wherein CD11c + CD11b + is a Ly6c + DC cell subset and CD11c-CD11b + is an M-MDSC cell subset;
within the CD19-Ly6 c-cell subset, a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is a myeloid DC cell subset and CD11c + CD11b + is a plasma DC cell subset.
In a third aspect, the invention provides the kit for simultaneously detecting multiple cell subsets and their functional changes according to the first aspect and/or the method for detecting changes in immune cell subsets according to the second aspect, and uses thereof in the detection of functional changes in immune cell subsets.
Compared with the prior art, the invention has the following beneficial effects:
the antibody composition covers markers of multiple immune cells, can simultaneously detect the change conditions of the immune levels of multiple cell subsets in one-time flow analysis, and has specific and comprehensive results and higher application value; the kit for simultaneously detecting multiple cell subsets is convenient to use and easy to operate; by matching with a method for detecting the change of immune cell subsets, the change difference of various immune cell subsets including CD 4T cells, CD 8T cells, Treg cells, granulocytes, monocytes, macrophages, B cells, NK cells, MDSC cells and DC cells can be simultaneously detected in one detection reaction, and the method is used for analyzing an immune map, has important significance for the evaluation of the immune level of an organism, the judgment of disease process, the evaluation of treatment effect and the screening of prognosis indexes, and has wide application prospect.
Drawings
Fig. 1A is a picture of the result of the flow-type detection in example 3 of the present invention, in which a picture I is a picture of the result of the direct detection, and a picture II is a picture of the result of the detection after cell debris is removed; FIG. III is a photograph showing the results of detection after removal of adherent cells;
FIG. 1B is a photograph showing the cell distribution of the CD45+ cytogate in example 3 of the present invention;
FIG. 1C is a photograph showing the cell distribution of the Ly6G + cell subset in example 3 of the present invention;
FIG. 1D is a photograph showing the cell distribution of Ly 6G-cell subset in example 3 of the present invention;
FIG. 1E is a photograph showing the cell distribution of NK + cell subsets in example 3 of the present invention;
FIG. 1F is a photograph showing the cell distribution of F4/80-NK 1.1-cell subset in example 3 of the present invention;
FIG. 1G is a photograph showing the cell distribution of a CD3/SSC cell gate set in example 3 of the present invention, wherein FIG. I is a photograph showing the cell distribution of a CD3+ cell subset, and FIG. II is a photograph showing the cell distribution of a CD 3-cell subset;
FIG. 1H is a picture of cell distribution of CD8+ T cell subsets in example 3 of the present invention, in which FIG. I is a picture of expression of Granzyme B, FIG. II is a picture of expression of Perforin, and FIG. III is a picture of expression of IFN- γ;
FIG. 1I is a photograph of the cell distribution of the CD4+ T cell subset in example 3 of the present invention;
fig. 1J is a picture of the cell distribution of the Tregs cell subpopulation in example 3 of the present invention;
FIG. 1K is a photograph showing the cell distribution of a subpopulation of CD19-Ly6c + cells in example 3 of the present invention;
FIG. 1L is a photograph showing the cell distribution of CD19-Ly6 c-cell subset in example 3 of the present invention;
fig. 2A is a picture of a result of the streaming detection in example 4 of the present invention, in which a picture I is a picture of a direct detection result, and a picture II is a picture of a detection result after cell debris is removed; FIG. III is a photograph showing the results of detection after removal of adherent cells;
FIG. 2B is a photograph showing the cell distribution of the CD45+ cytogate in example 4 of the present invention;
FIG. 2C is a photograph showing the cell distribution of the Ly6G + cell subset in example 4 of the present invention;
FIG. 2D is a photograph showing the cell distribution of Ly 6G-cell subset in example 4 of the present invention, wherein FIG. I is a photograph showing the cell distribution setting of the F4/80/NK1.1 cell gate, and FIG. II is a photograph showing the cell distribution setting of the F4/80/CD11b cell gate;
FIG. 2E is a photograph showing the cell distribution of F4/80+ CD11b + cell subset in example 4 of the present invention;
FIG. 2F is a photograph of the cell distribution of NK + cell subsets in example 4 of the present invention;
FIG. 2G is a photograph showing the cell distribution of F4/80-NK 1.1-cell subset in example 4 of the present invention;
FIG. 2H is a photograph showing the cell distribution of a CD3/SSC cell gate set in example 4 of the present invention, wherein FIG. I is a photograph showing the cell distribution of a CD3+ cell subset, and FIG. II is a photograph showing the cell distribution of a CD 3-cell subset;
FIG. 2I is a picture of cell distribution of CD8+ T cell subsets in example 4 of the present invention, wherein I is a picture of expression of Granzyme B, II is a picture of expression of Perforin, and III is a picture of expression of IFN- γ;
FIG. 2J is a photograph of the cell distribution of the CD4+ T cell subset in example 4 of the present invention;
FIG. 2K is a graph of the cell distribution of the Tregs cell subpopulation in example 4 of the present invention;
FIG. 2L is a photograph showing the cell distribution of a subpopulation of CD19-Ly6c + cells in example 4 of the present invention;
FIG. 2M is a photograph showing the cell distribution of CD19-Ly6 c-cell subset in example 4 of the present invention;
fig. 3A is a picture of a result of the flow-type detection in example 5 of the present invention, in which a picture I is a picture of a direct detection result, and a picture II is a picture of a detection result after cell debris is removed; FIG. III is a photograph showing the results of detection after removal of adherent cells;
FIG. 3B is a photograph showing the cell distribution of the CD45+ cytogate in example 5 of the present invention;
FIG. 3C is a photograph showing the cell distribution of the Ly6G + cell subset in example 5 of the present invention;
FIG. 3D is a photograph showing the cell distribution of Ly 6G-cell subset in example 5, wherein FIG. I is a photograph showing the cell distribution setting of the F4/80/NK1.1 cell gate, and FIG. II is a photograph showing the cell distribution setting of the F4/80/CD11b cell gate;
FIG. 3E is a photograph showing the cell distribution of F4/80+ CD11b + cell subset in example 5 of the present invention;
FIG. 3F is a photograph showing the cell distribution of F4/80-NK 1.1-cell subset in example 5 of the present invention;
FIG. 3G is a photograph showing the cell distribution of a CD3/SSC cell gate set in example 5 of the present invention, in which FIG. I is a photograph showing the cell distribution of a CD3+ cell subset, and FIG. II is a photograph showing the cell distribution of a CD 3-cell subset;
FIG. 3H is a picture of cell distribution of CD8+ T cell subsets in example 5 of the present invention, in which FIG. I is a picture of expression of Granzyme B, FIG. II is a picture of expression of Perforin, and FIG. III is a picture of expression of IFN-. gamma.;
FIG. 3I is a photograph of the cell distribution of the CD4+ T cell subset in example 5 of the present invention;
FIG. 3J is a photograph of the cell distribution of the Tregs cell subpopulation in example 5 of the present invention;
FIG. 3K is a photograph showing the cell distribution of a subpopulation of CD19-Ly6c + cells in example 5 of the present invention;
FIG. 3L is a photograph showing the cell distribution of CD19-Ly6 c-cell subset in example 5 of the present invention.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Materials:
zombie NIR was purchased from Biolegend, cat # 423106;
CD45 was purchased from eBioscience, cat # 69-0451-82;
ly6G was purchased from Biolegend, cat No. 127616;
CD11b was purchased from Biolegend, cat # 101262;
f4/80 was purchased from Biolegend, cat # 123133;
ly6c was purchased from Biolegend, cat No. 128044;
NK1.1 purchased from Biolegend, cat # 108730;
CD3 was purchased from Biolegend, cat # 100210;
CD4 was purchased from Biolegend, cat # 100555;
CD25 was purchased from Biolegend, cat # 102049;
FoxP3 was purchased from Biolegend, cat No. 126404;
IL-10 was purchased from Biolegend, cat # 505021;
CD8a was purchased from Biolegend, cat # 100740;
granzyme B was purchased from eBioscience, cat # 46-8898-82;
perforin is available from Biolegend, cat # 154304;
IFN- γ was purchased from Biolegend, cat # 505838;
CD19 was purchased from Biolegend, cat # 115523;
CD11c was purchased from Biolegend, cat # 117357;
CD80 was purchased from Biolegend, cat # 104712;
CD206 was purchased from Biolegend, cat # 141720;
ACK red blood cell lysates were purchased from Biolegend, cat # 420301;
RPMI-1640 medium was purchased from GibcoTM A1049101
Blocking agents were purchased from Biolegend, cat # 101320;
antibody buffer was purchased from BD, cat # 7324799;
fixative was purchased from Biolegend, cat # 420801;
rupture fluid was purchased from Biolegend, cat # 421002;
flow cytometry was purchased from BD, cat # 8218730.
Example 1
This example provides an antibody composition comprising a surface antibody composition and an intrabody composition.
The surface antibody composition comprises a combination of at least two of Zombie NIR, CD45, Ly6G, CD11b, F4/80, Ly6c, NK1.1, CD3, CD4, CD25, CD8a, CD19, CD11c, CD80, or CD 206;
the intrabody composition includes a combination of at least two of FoxP3, IL-10, Granzyme B, Perforin, or IFN- γ.
The antibody composition comprises, in parts by weight, Zombie NIR 0.1 part, CD 450.3 part, Ly6G 0.3 part, CD11B 0.3 part, F4/800.6 part, Ly6c 0.08.08 part, NK1.10.5 part, CD 30.2 part, CD 40.15 part, CD 250.1 part, CD8a 0.3 part, CD 190.2 part, CD11c 1 part, CD 802 part, CD 2061 part, FoxP 30.5 part, IL-102.5 part, Granzyme B0.3 part, Perforin 5 part and IFN-gamma 5 part.
The antibody composition disclosed by the invention covers markers of various immune cells, and can be used for combined detection of variation difference of immune levels of various cell subsets.
Example 2
This example provides a kit for simultaneously detecting multiple cell subsets comprising the antibody composition of example 1;
the kit for simultaneously detecting multiple cell subsets further comprises a sealing agent, a membrane-breaking liquid and a flow cytometry liquid.
Example 3
This example uses the kit for simultaneously detecting multiple cell subsets in example 2 to detect the change of the subpopulation of immune cells in peripheral blood of mice, and includes the following steps:
(one) preparation of Single cell suspensions
1. Immediately taking 200 mul of fresh blood to an EP tube containing 200 mul of 3.8% sodium citrate anticoagulant, mixing uniformly, and centrifuging at 4 ℃ and 1500g for 15 min;
2. adding 1mL PBS into blood cells, resuspending and washing, centrifuging at 4 ℃ and 350g for 5min, and removing supernatant;
adding 3mL of ACK erythrocyte lysate into each tube of a 3.15 mL centrifuge tube, gently blowing, beating and shaking, and performing lysis on ice for 2 min;
4. adding HBSS solution 2 times the volume of HBSS solution, mixing, centrifuging at 4 deg.C and 350g for 5min, and removing supernatant;
5. resuspend the cell pellet.
(II) blocking with blocking agent, staining with surface antibody composition, washing, and resuspending the cells
1. Adding 1 μ L of blocking agent, and incubating on ice in dark for 10 min;
2. adding the surface antibody composition and 20 μ L antibody buffer solution, mixing, and incubating in dark for 30 min;
centrifuging at 400g for 5min at 3.4 deg.C, and removing supernatant;
4. the cells were washed with 0.8mL PBS, mixed well, centrifuged at 500g for 5min, the supernatant was discarded and resuspended in 100. mu.L PBS.
(III) Membrane rupture treatment, adding an intracellular antibody composition
1. Adding 1mL of membrane breaking liquid into the tube, and incubating for 60min at 4 ℃ in a dark place;
centrifuging at 2.500 g for 5min, discarding the supernatant, and resuspending the cells with 100 μ L of membrane-breaking solution;
3. adding the intracellular antibody composition, incubating at room temperature in the dark for 30min, adding 1mL of membrane breaking solution, centrifuging at 500g for 5min, and discarding the supernatant;
4. then 1mL of membrane breaking solution is added, 500g of the solution is centrifuged for 5min, and the supernatant is discarded.
(IV) resuspending the cell fluid by using a flow type cell fluid, detecting the cell fluid on a machine, and analyzing the cell fluid
The analysis comprises the following steps:
1. the flow assay results are shown in panel I of FIG. 1A, with cell debris removed from the cell gate, as shown in panel II of FIG. 1A, and adherent cells removed, as shown in panel III of FIG. 1A;
2. the CD45+ cell gate was set, and as a result, as shown in FIG. 1B, multiple gates were set to detect the expression of multiple cell populations:
3. setting the Ly6G/SSC cell gate, dividing into Ly6G + cell subset (FIG. 1C) and Ly 6G-cell subset (FIG. 1D);
(1) within the Ly6G + cell subset (fig. 1C), the Ly6C/CD11b phyla was set, where CD11b + is neutrophils and Ly6C divides neutrophils into two subsets, Ly6C + and Ly6C-, the Ly6C + subset being the PMN-MDSC cell population;
(2) within Ly 6G-cell subset (FIG. 1D), F4/80/NK1.1 cell gate was set, dividing the cells into four cell subsets F4/80+ NK1.1+, F4/80+ NK1.1-, F4/80-NK1.1+ and F4/80-NK 1.1-;
1) F4/80-NK1.1+ is NK + cell subset (FIG. 1E), setting CD3/SSC cell gate, and dividing into CD3+ NK T cell subset and NK cell subset;
2) within the F4/80-NK 1.1-cell subset (FIG. 1F), a CD3/SSC cell gate was set, divided into a CD3+ cell subset (FIG. 1G, panel I) and a CD 3-cell subset (FIG. 1G, panel II);
setting a CD4/CD8 cell gate in a CD3+ T cell subgroup (figure 1G, figure I), and dividing the cell gate into three cell subgroups of CD8+ T cells, CD4+ T cells and DNT cells;
analyzing the expression of IFN-gamma (FIG. 1H, panel III), Perforin (FIG. 1H, panel II) and Granzyme B (FIG. 1H, panel I) within the CD8+ T cell subset;
setting a CD25/FoxP3 cell gate in a CD4+ T cell subset (figure 1I), wherein CD25+ FoxP3+ is a Tregs cell subset (figure 1J), and further analyzing the expression condition of IL-10 in the Tregs cell subset (figure 1J);
② within CD 3-cell subsets (FIG. 1G, II), setting Ly6c/CD19 cell gate, wherein CD19+ Ly6 c-is B cell subset;
within the CD19-Ly6c + cell subset (FIG. 1K), a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is the Ly6c + DC cell subset and CD11c-CD11b + is the M-MDSC cell subset;
within the CD19-Ly6 c-cell subset (FIG. 1L), a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is a myeloid DC cell subset and CD11c + CD11b + is a plasma DC cell subset.
Example 4
This example uses the kit for simultaneously detecting multiple cell subsets of example 2 to detect the change of the subset of immune cells in mouse spleen, and comprises the following steps:
(one) preparation of Single cell suspensions
1. Placing the spleen into a sterile 10cm culture dish containing 5mL of RPMI-1640 culture medium, cutting the spleen into small pieces, and lightly grinding the spleen by using a syringe piston to enable dispersed single cells to enter the culture medium through a nylon net;
2. placing a sterilized 200-mesh screen on a 15mL centrifuge tube, collecting cells into the centrifuge tube, placing on ice, centrifuging at 4 ℃ under 350g for 5min, removing supernatant, and slightly bouncing cell sediment;
3. adding RPMI-1640 culture medium into a centrifuge tube until the final volume is 15mL, centrifuging at 4 ℃ and 350g for 5min, and discarding the supernatant;
4. adding the mixture into 5mL of ACK erythrocyte lysate, slowly shaking to resuspend the cells and accelerate the lysis of the erythrocytes;
after 5.2min, adding RPMI-1640 culture medium to 15mL, terminating the lysis, centrifuging at 4 ℃ under 350g for 5min, and discarding the supernatant;
6. adding 12mL of PBS, gently blowing to resuspend the cells, standing for 2min, taking 10mL of cell suspension at the upper part, and transferring the cell suspension into another 15mL centrifuge tube;
7. viable cells were counted using trypan blue and the concentration was adjusted to 2X 106/mL。
(II) after blocking with blocking agent, staining with surface antibody composition, and after washing, resuspending the cells as in example 3.
(III) Membrane rupture treatment, adding an intracellular antibody composition
Same as in example 3.
(IV) resuspending the cell fluid by using a flow type cell fluid, detecting the cell fluid on a machine, and analyzing the cell fluid
The analysis comprises the following steps:
1. the flow assay results are shown in panel I of fig. 2A, with cell debris removed from the cell gate, as shown in panel II of fig. 2A, and adherent cells removed, as shown in panel III of fig. 2A;
2. the results of setting the CD45+ cell gate are shown in FIG. 2B, and setting multiple gates to detect the expression of multiple groups of cells:
3. setting Ly6G/SSC cell gate, dividing into Ly6G + cell subset (FIG. 2C) and Ly 6G-cell subset (FIG. 2D, panels I and II);
(1) within the Ly6G + cell subset (fig. 2C), the Ly6C/CD11b phyla was set, where CD11b + is neutrophils and Ly6C divides neutrophils into two subsets, Ly6C + and Ly6C-, the Ly6C + subset being the PMN-MDSC cell population;
(2) setting a F4/80/CD11b cytogate in Ly 6G-cytosubgroup (figure 2D, figure II), and dividing the cytoblast into four cytosubgroups of F4/80+ CD11b +, F4/80+ CD11b-, F4/80-CD11b + and F4/80-CD11 b-;
within the F4/80+ CD11b + cell subset (fig. 2E), a CD80/CD206 cell gate was set, wherein CD80+ CD206 "is macrophage subset M1 and CD80-CD206+ is macrophage subset M2;
(3) within Ly 6G-cell subset (FIG. 2D, I), F4/80/NK1.1 cell gate was set to divide the cells into four cell subsets F4/80+ NK1.1+, F4/80+ NK1.1-, F4/80-NK1.1+ and F4/80-NK 1.1-;
1) F4/80-NK1.1+ is NK + cell subset (FIG. 2F), setting CD3/SSC cell gate, and dividing into CD3+ NK T cell subset and NK cell subset;
2) within the F4/80-NK 1.1-cell subset (FIG. 2G), a CD3/SSC cell gate was set, divided into a CD3+ cell subset (FIG. 2H, panel I) and a CD 3-cell subset (FIG. 2H, panel II);
setting a CD4/CD8 cell gate in a CD3+ T cell subgroup (figure 2H and figure I), and dividing the cell gate into three cell subgroups of CD8+ T cells, CD4+ T cells and DNT cells;
analyzing the expression of IFN-gamma (FIG. 2I, panel III), Perforin (FIG. 2I, panel II) and Granzyme B (FIG. 2I, panel I) within the CD8+ T cell subpopulation;
setting a CD25/FoxP3 cell gate in a CD4+ T cell subset (figure 2J), wherein CD25+ FoxP3+ is a Tregs cell subset (figure 2K), and further analyzing the expression condition of IL-10 in the Tregs cell subset (figure 2K);
② within CD 3-cell subsets (FIG. 2H, panel II), setting Ly6c/CD19 cell gate, wherein CD19+ Ly6 c-is B cell subset;
within the CD19-Ly6c + cell subset (FIG. 2L), a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is the Ly6c + DC cell subset and CD11c-CD11b + is the M-MDSC cell subset;
within the CD19-Ly6 c-cell subset (FIG. 2M), a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is a myeloid DC cell subset and CD11c + CD11b + is a plasma DC cell subset.
Example 5
This example uses the kit for simultaneously detecting multiple cell subsets of example 2 to detect the change of the subpopulation of immune cells of mouse tumor, and comprises the following steps:
(one) preparation of Single cell suspensions
1. Adding 3mL of precooled HBSS buffer solution into a 6cm culture dish, placing the tumor on a stainless steel screen mesh of 100 meshes, cutting the tumor tissue into small pieces by using a scalpel, and grinding the tumor tissue by using an injector piston to disperse cells into the culture dish solution;
2. placing a 200-mesh screen on a 15mL centrifuge tube, collecting the cell suspension into the centrifuge tube through the screen, placing the centrifuge tube on ice, centrifuging the centrifuge tube for 5min at the temperature of 4 ℃ and the temperature of 350g, and removing supernatant;
3. resuspend the cell pellet with 1mL of ACK lysis for 1min, lyse the red blood cells, and resuspend the cells with HBSS buffer.
(II) after blocking with blocking agent, staining with surface antibody composition, and after washing, resuspending the cells as in example 3.
(III) Membrane rupture treatment, adding an intracellular antibody composition
Same as in example 3.
(IV) resuspending the cell fluid by using a flow type cell fluid, detecting the cell fluid on a machine, and analyzing the cell fluid
The analysis comprises the following steps:
1. the flow assay results are shown in panel I of fig. 3A, with cell debris removed from the cell gate, as shown in panel II of fig. 3A, and adherent cells removed, as shown in panel III of fig. 3A;
2. the results of setting the CD45+ cell gate are shown in FIG. 3B, and setting multiple gates to detect the expression of multiple groups of cells:
3. setting Ly6G/SSC cell gate, dividing into Ly6G + cell subset (FIG. 3C) and Ly 6G-cell subset (FIG. 3D, panels I and II);
(1) within the Ly6G + cell subset (fig. 3C), the Ly6C/CD11b phyla was set, where CD11b + is neutrophils and Ly6C divides neutrophils into two subsets, Ly6C + and Ly6C-, the Ly6C + subset being the PMN-MDSC cell population;
(2) setting a F4/80/CD11b cytogate in Ly 6G-cytosubgroup (figure 3D, figure II), and dividing the cytoblast into four cytosubgroups of F4/80+ CD11b +, F4/80+ CD11b-, F4/80-CD11b + and F4/80-CD11 b-;
within the F4/80+ CD11b + cell subset (fig. 3E), a CD80/CD206 cell gate was set, wherein CD80+ CD206 "is macrophage subset M1 and CD80-CD206+ is macrophage subset M2;
(3) within Ly 6G-cell subset (FIG. 3D, I), F4/80/NK1.1 cell gate was set to divide the cells into four cell subsets F4/80+ NK1.1+, F4/80+ NK1.1-, F4/80-NK1.1+ and F4/80-NK 1.1-;
1) within the F4/80-NK 1.1-cell subset (FIG. 3F), a CD3/SSC cell gate was set, divided into a CD3+ cell subset (FIG. 3G, panel I) and a CD 3-cell subset (FIG. 3G, panel II);
setting a CD4/CD8 cell gate in a CD3+ T cell subgroup (figure 3G, figure I), and dividing the cell gate into three cell subgroups of CD8+ T cells, CD4+ T cells and DNT cells;
analyzing the expression of IFN-gamma (FIG. 3H, panel III), Perforin (FIG. 3H, panel II) and Granzyme B (FIG. 3H, panel I) within the CD8+ T cell subpopulation;
setting a CD25/FoxP3 cell gate in a CD4+ T cell subset (figure 3I), wherein CD25+ FoxP3+ is a Tregs cell subset (figure 3J), and further analyzing the expression condition of IL-10 in the Tregs cell subset (figure 3J);
② within CD 3-cell subsets (FIG. 3G, II), setting Ly6c/CD19 cell gate, wherein CD19+ Ly6 c-is B cell subset;
within the CD19-Ly6c + cell subset (FIG. 3K), a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is the Ly6c + DC cell subset and CD11c-CD11b + is the M-MDSC cell subset;
within the CD19-Ly6 c-cell subset (FIG. 3L), a CD11cCD11b cell gate was set, wherein CD11c + CD11b + is a myeloid DC cell subset and CD11c + CD11b + is a plasma DC cell subset.
By combining the results of the embodiments 3-5, it can be seen that the kit for simultaneously detecting multiple cell subsets, provided by the invention, and matched with a corresponding detection method, can simultaneously detect the variation difference of multiple immune cell subsets in one detection reaction, is suitable for immune cells in multiple tissues, has more specific and accurate results, and has practical application value.
In conclusion, the antibody composition covers various markers of immune cells, can simultaneously detect and analyze the change difference of the immune cells in different organs in one detection reaction by virtue of a flow analysis technology, and has important significance for judging the disease process, screening a treatment method and judging the treatment effect; simple operation and high detection efficiency, and has wide application prospect in the mechanism research and clinical practice of diseases.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (10)

1. A kit for simultaneously detecting multiple cell subsets and functional changes is characterized in that the kit for simultaneously detecting multiple cell subsets and functional changes comprises an antibody composition;
the antibody compositions include surface antibody compositions and intrabody compositions;
the surface antibody composition comprises a combination of at least two of Zombie NIR, CD45, Ly6G, CD11b, F4/80, Ly6c, NK1.1, CD3, CD4, CD25, CD8a, CD19, CD11c, CD80, or CD 206;
the intrabody composition includes a combination of at least two of FoxP3, IL-10, Granzyme B, Perforin, or IFN- γ.
2. The kit for simultaneously detecting multiple cell subsets and functional changes according to claim 1, wherein the antibody composition comprises, in parts by weight: 0.05-0.2 part of Zombie NIR, 450.2-0.5 part of CD, 0.2-0.5 part of Ly6G 0.2, 0.2-0.5 part of CD11B 0.2, 0.2-0.5 part of F4/800.5-0.7 part of Ly6c 0.05, 0.05-0.1 part of NK1.10.1-0.8 part of CD 30.1-0.5 part of CD 40.1-0.3 part of CD 250.05-0.3 part of CD8a 0.1, 0.1-0.5 part of CD 190.1-0.5 part of CD11c 0.5, 0.5-3 part of CD 801, 4-3 parts of CD XP 2060.5-3 parts of FoxP30.3-0.8 part of IL-102-5 parts of Granzyme B, 0.1-0.5 part of Perforin and 3-8 parts of IFN-gamma.
3. The kit for simultaneously detecting multiple cell subsets and functional changes according to claim 1 or 2, wherein the antibody composition comprises, in parts by weight: 0.08-0.15 part of Zombie NIR, 450.2-0.4 part of CD, 0.2-0.4 part of Ly6G 0.2, 0.2-0.4 part of CD11B 0.2, 0.2-0.4 part of F4/800.5-0.65 part of Ly6c 0.06, 0.06-0.1 part of NK1.10.4-0.7 part of CD 30.1-0.4 part of CD 40.1-0.2 part of CD 250.05-0.2 part of CD8a 0.1, 0.1-0.4 part of CD 190.1-0.4 part of CD11c 0.5, 0.5-2 part of CD 801, 3-2 parts of CD XP 2060.5, 0.3-0.6 part of Fonze B, 3-6 parts of IL-102-4 parts of Granze B, 3-6 parts of Perforin and 3-6 parts of IFN-gamma.
4. The kit for simultaneously detecting multiple cell subsets and functional changes according to any one of claims 1-3, wherein the kit for simultaneously detecting multiple cell subsets and functional changes further comprises a blocking agent, a membrane disruption agent and an on-machine buffer.
5. The kit for simultaneously detecting multiple cell subsets and functional changes according to claims 1-4, wherein the cells comprise any one or a combination of at least two of CD 4T cells, CD 8T cells, Treg cells, double negative T cells, granulocytes, macrophages, B cells, NK cells, MDSC cells, or DC cells.
6. A method for detecting changes in immune cell subsets, which comprises using the kit for simultaneously detecting multiple cell subsets and functional changes according to any one of claims 1 to 5.
7. The method of detecting a change in a subpopulation of immune cells according to claim 6, wherein said method comprises:
preparing single cell suspension, sealing with sealant, staining with surface antibody composition, washing, re-suspending cells, treating with membrane-breaking solution, adding intracellular antibody composition, incubating, removing supernatant, re-suspending with on-machine buffer solution, detecting on-machine, and analyzing.
8. The method according to claim 7, wherein the flow cytometer used in the on-machine detection is a full spectrum flow cytometer;
preferably, the analysis comprises:
removing cell debris from the cell compartment and then adherent cells;
setting multiple cell gates, and detecting the expression of multiple cell subsets.
9. The method for detecting a change in a subpopulation of immune cells according to any one of claims 6 to 8, wherein said method for detecting a change in a subpopulation of immune cells comprises:
preparing single cell suspension, sealing with a sealing agent, staining with a surface antibody composition, washing, resuspending cells, adding a membrane breaking solution for treatment, adding an intracellular antibody composition, incubating, removing supernatant, resuspending with an on-machine buffer solution, performing on-machine detection with a full-spectrum flow cytometer, and analyzing;
the analysis comprises the following steps:
removing cell debris from the cell compartment and then adherent cells;
setting multiple cell gates, and detecting the expression of multiple cell subsets.
10. Use of the kit for simultaneous detection of multiple cell subsets and functional changes according to any of claims 1-5 and/or the method for detecting changes in immune cell subsets according to any of claims 6-9 for the detection of functional changes in immune cell subsets.
CN202111164758.5A 2021-09-30 2021-09-30 Kit for simultaneously detecting various cell subsets and functional changes and application thereof Pending CN113866409A (en)

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