CN117783532A

CN117783532A - Kit for detecting human blood follicular T helper cell subtype and application thereof

Info

Publication number: CN117783532A
Application number: CN202310697910.9A
Authority: CN
Inventors: 王君灵; 秦秉玉; 湛萌萌; 董子宜
Original assignee: Henan Provincial Peoples Hospital
Current assignee: Henan Provincial Peoples Hospital
Priority date: 2023-06-13
Filing date: 2023-06-13
Publication date: 2024-03-29

Abstract

The invention belongs to the technical field of flow cytometry detection, and particularly relates to a kit for detecting human blood follicular T helper cell subtype and application thereof. The T helper cell of the blood follicular is CD3 ⁺ CD4 ^‑ CD8 ⁺ T helper cells, the kit comprising a blood dilution, a mononuclear cell separation, a cell culture, a dead cell removal dye, an FcR blocker solution, a fluorescently labeled antibody against a human cell membrane surface molecule, a wash buffer. The kit and the detection method can be used for simply and rapidly analyzing the follicular CD3 ⁺ CD4 ^‑ CD8 ⁺ T helper cells and subtypes thereof, and may be based on CD3 ⁺ CD4 ^‑ CD8 ⁺ The proportion of T helper cells and subtypes aids in the clinical diagnosis of inflammatory diseases such as allergies and sepsis.

Description

Kit for detecting human blood follicular T helper cell subtype and application thereof

Technical Field

The invention belongs to the technical field of flow cytometry detection, and particularly relates to a kit for detecting human blood follicular T helper cell subtype and application thereof.

Background

Early studies found that T follicular helper cells (T follicle helper cells, tfh cells) were C in the B cell follicular centerD4 ⁺ A specific subset of T cells induces B cell interactions with T cells and secretion of cytokines that promote follicular germinal center formation, promote germinal center B cell differentiation into memory B cells or plasma cells, and induce plasma cells to synthesize and secrete high affinity antibodies. However, human peripheral blood CD4 was not found until 1994 ⁺ CXCR5 ⁺ Tfh. Later, the scholars of Rimpei Morita et al in 2011 defined human peripheral blood Tfh cells as three subpopulations, i.e., pairs, based on whether Tfh expressed CXCR3 and CCR6CD4 without significant effect on B cell differentiation and development ⁺ CXCR5 ⁺ CXCR3 ⁺ CCR6 ^- Tfh1, can secrete IL-21 and induce +.>Differentiation and development of B cells into memory B cells and plasma cells to promote antibody production by plasma cells ⁺ CXCR5 ⁺ CXCR3 ^- CCR6 ^- Tfh2 and CD4 ⁺ CXCR5 ⁺ CXCR3 ^- CC R6 ⁺ Tfh17 cells.

Notably, schnikoogs, chenYuhong et al, in 2019, first discovered that Stat5 negatively regulated CD8 in mouse spleen follicles ⁺ CXCR5 ⁺ PD1 ⁺ Tfh cells. CD8 as described above ⁺ Tfh cells regulate germinal center B cell responses and control autoantibody production, and CD4 ⁺ Tfh has similar genetic characteristics and requires CD40L/CD40 and TCR/mhc i interactions to assist B cells. In addition, murayama K et al, in 2022, found a large number of CD3 at the center of the germinal area at the site of fiber inflammation in patients with IgG 4-related diseases ⁺ CD4 ⁺ CD8 ⁺ CXCR5 ⁺⁺ PD1 ⁺⁺ Tfh cells. In summary, the existing studies have focused mainly on mouse spleen CD8 ⁺ Tfh cells and human fibrotic tissue CD4 ⁺ CD8 ⁺ Tfh cells. Currently, the existing kit for detecting human blood follicular T helper cell subtype detects CD3 ⁺ CD4 ⁺ Tfh cells (e.g. proprietaryNumber of interest: CN109406775 a), but the presence of such a kit did not confirm CD3 ⁺ CD4 ⁺ Tfh cells are a problem in blood. Therefore, such a kit is not suitable for detecting T-follicular helper cells in human peripheral blood. At present, human peripheral blood CD3 is not detected ⁺ CD4 ^- CD8 ⁺ Tfh cells and a kit for a subtype thereof. Therefore, development of a kit capable of detecting CD3 in human peripheral blood is desired ⁺ CD4 ^- CD8 ⁺ The Tfh cells and the kit of the subtype thereof can be used for diagnosing allergic diseases and sepsis.

Disclosure of Invention

To solve the problem that the prior art is not used for detecting CD3 of human peripheral blood ⁺ CD4 ^- CD8 ⁺ The invention provides a kit for detecting human blood follicular T helper cell subtype for the first time, and the kit utilizes flow cytometry to identify human peripheral blood CD3 ⁺ CD4 ^- CD8 ⁺ T helper cells and subtypes thereof, the following technical scheme is adopted:

the invention provides a kit for detecting subtype of human blood follicular T auxiliary cells, wherein the blood follicular T auxiliary cells are CD3 ⁺ CD4 ^- CD8 ⁺ Tfh cells, the kit comprises blood dilutions, mononuclear cell separation, cell culture, dead cell removal dye, fcR blocker solution, fluorescently labeled antibodies against human cell membrane surface molecules, and wash buffer.

Preferably, the blood dilution comprises a NaCl solution or a PBS buffer; the mass concentration of the NaCl solution is 0.85-0.95%;

the PBS buffer solution comprises the following components in parts by weight: 7.8 to 8.2 parts of NaCl,0.15 to 0.25 part of KCl and 1.14 to 1.18 parts of Na ₂ HPO ₄ And 0.15 to 0.25 part KH ₂ PO ₄ ；

The penetration concentration of the PBS is 280-320 mMol/L;

the pH value of the PBS buffer solution is 7.0-7.6.

Preferably, the mononuclear cell separation liquid has a density of 1.076-1.078 g/mL and an osmotic pressure of 275-305 mOsm.

Preferably, the formula of the cell culture fluid is as follows: RPMI 1640 is used as a basic culture medium, and fetal bovine serum and penicillin-streptomycin solution are added into the RPMI 1640; the final concentration of the fetal bovine serum is 0.5-15%; the final concentration of the penicillin-streptomycin solution is 0.5-2%.

Preferably, the dead cell removal dye is an amine reactive fluorescent dye.

Preferably, the FcR blocker solution comprises human immunoglobulins or unrelated immunoglobulins of the same genus and subtype as the streaming antibody used.

Preferably, the fluorescent species of the dead cell removal dye and fluorescently labeled antibody against human cell membrane surface molecules include fluorescence of one or more of Alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, alexa Fluor700, APC/Cy7, APC/H7, brilliant Violet421, brilliant Violet510, brilliant Blue515, brilliant Violet 570, brilliant Violet 605, brilliant Violet 650, brilliant Violet711, brilliant Violet 785, FITC, LEAF, pacific Blue, PE/Cy5, PE/dazle, perCP or PerCP/Cy 5.5.

Preferably, the fluorescently labeled antibody against a human cell membrane surface molecule comprises an anti-human CD3 epsilon antibody, an anti-human CD4 antibody, an anti-human CXCR5 antibody, an anti-human CCR6 antibody, and an anti-human CXCR3 antibody; or anti-human CD3 epsilon antibodies, anti-human CD4 antibodies, anti-human CD8 antibodies, anti-human CXCR5 antibodies, anti-human CCR6 antibodies, and anti-human CXCR3 antibodies.

The invention also provides application of the kit for detecting the subtype of the human blood follicular T auxiliary cells in diagnosis of allergic diseases and sepsis.

The invention also provides a method for detecting the subtype of the human blood follicular T auxiliary cell by using the kit for detecting the subtype of the human blood follicular T auxiliary cell, which comprises the following steps:

s1, separating mononuclear cells from peripheral venous blood of a human body;

s2, diluting the mononuclear cells obtained in the step S1 by using blood diluent, centrifuging, and collecting cell sediment;

s3, re-suspending the cell pellet obtained in S2 by using a washing buffer solution to obtain a cell concentration of 10 ⁶ ～10 ⁷ A first cell suspension of a single/mL;

s4, mixing the first cell suspension obtained in the S3 with a DMSO-dissolved dead cell removal dye and FcR blocker solution, and incubating at 4-40 ℃ for 5-30 min to obtain first incubated cells; mixing the first incubated cells with cell staining buffer, centrifuging, removing supernatant, and precipitating cells according to 10 ⁵ ～10 ⁶ Adding 0.5-20 mu L of fluorescent labeled antibody against human cell membrane surface molecules into each cell/100 mu L system, uniformly mixing, and then incubating for 5-30 min at 4-40 ℃ in a dark place to obtain second incubated cells;

s5, adding the second incubated cells obtained in the step S4 into a washing buffer, uniformly mixing, centrifuging, discarding the supernatant, and adjusting the cell concentration to 10 by using the washing buffer ⁵ ～10 ⁶ Detecting the expression of each antibody by a flow cytometer after each cell/mL; calculation of CD3 based on the expression of each antibody ⁺ CD4 ^- CD8 ⁺ Detection of Tfh cells and subtypes thereof.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for detecting human CD3 ⁺ CD4 ^- CD8 ⁺ Tfh cells and its subtype kit, and include its detection method and application in diagnosis of allergic diseases and sepsis. The reagent and the raw materials in the kit are simple and easy to obtain, and the reagent is matched with each other to play a role in detection. The kit comprises blood diluent, mononuclear cell separating liquid, cell culture liquid, dead cell removing dye, fcR blocker solution, fluorescent labeled antibody against human cell membrane surface molecules and washing buffer solution, and the detection method is rapid and convenient: after preparing human peripheral blood mononuclear, adding dead cell removal dye and FcR blocker, and then performing fluorescent antibody staining, and after washing cells, detecting by using a flow cytometer, and can be used for auxiliary diagnosis of allergic diseases and sepsis.

Drawings

FIG. 1 is a schematic representation of the present invention for flow cytometry identification of CD3 ⁺ CD4 ^- CD8 ⁺ Schematic flow sheet of Tfh cells and subtypes thereof;

FIG. 2 is a flow-through representative diagram of human peripheral blood mononuclear cells (A), human peripheral blood mononuclear cells after removal of adherent cells (B), and human peripheral blood mononuclear cells after removal of dead cells (C);

FIG. 3 shows CD3 ε in peripheral blood mononuclear cells of healthy person (A), allergic asthma patient (B) and sepsis patient (C) ⁺ Flow-through representation of T cell expression levels, CD3 epsilon in peripheral blood of healthy person (D), allergic asthma patient (E) and sepsis patient (F) ⁺ CD4 in T cells ^- Flow-through representation of T cell expression levels, CD3 epsilon in peripheral blood of healthy person (G), allergic asthma patient (H) and sepsis patient (I) ⁺ CD4 ^- CXCR5 in T cells ⁺ Cell (CD 8) ⁺ Tfh) a flow-through representation of expression levels;

FIG. 4 is a graph of CD3 epsilon in peripheral blood of healthy person (A), allergic asthma patient (B) and sepsis patient (C) ⁺ CD4 ^- CXCR5 ⁺ T(CD8 ⁺ Tfh) CCR6 in cells ^- CXCR3 ⁺ (CD8 ⁺ Tfh 1) cells, CCR6 ^- CXCR3 ^- (CD8 ⁺ Tfh 2) cells and CCR6 ⁺ CXCR3 ^- (CD8 ⁺ Tfh 17) a flow-through representation of cell expression levels;

FIG. 5 shows CD3 ε in peripheral blood mononuclear cell populations of healthy people, allergic asthma patients and sepsis patients ⁺ CD4 ^- CD8 in T cells ⁺ Tfh cells (A), CD8 ⁺ Tfh1 cells (B), CD8 ⁺ Tfh2 cells (C) and CD8 ⁺ Statistical plots of Tfh17 cell (D) expression levels.

Detailed Description

The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.

The invention provides a kit for detecting human blood follicular T helper cell subtype, which comprisesThe T helper cell of blood follicular is CD3 ⁺ CD4 ^- CD8 ⁺ Tfh cells;

the kit comprises the following components: blood dilutions, mononuclear cell isolates, cell cultures, dead cell removal dyes, fcR blocker solutions, fluorescently labeled antibodies against human cell membrane surface molecules, wash buffers.

In the invention, the blood dilution liquid and the washing buffer liquid are preferably solutions with the osmotic pressure equal to that of human plasma, and the blood dilution liquid and the washing buffer liquid comprise NaCl solution or PBS buffer liquid, wherein the mass concentration of the NaCl solution is preferably 0.9%;

in the present invention, the PBS buffer preferably includes the following components in parts by weight: 8.0 parts of NaCl,0.20 parts of KCl and 1.16 parts of Na ₂ HPO ₄ And 0.20 part KH ₂ PO ₄ The method comprises the steps of carrying out a first treatment on the surface of the The pH of the PBS buffer is preferably 7.0 to 7.6, more preferably 7.2 to 7.5. The source of the blood dilution liquid is not particularly limited in the present invention.

In the present invention, the active ingredients of the mononuclear cell separation liquid preferably include: the solvent of the mononuclear cell separation liquid is preferably distilled water. The source and variety of the mononuclear cell separation liquid are not particularly limited, and the preferred mononuclear cell separation liquid has a density of 1.076-1.078 g/mL and an osmotic pressure of 275-305 mOsm.

In the present invention, the variety and source of the cell culture solution are not particularly limited, and a culture solution for culturing human blood cells is preferable, and the culture solution is more preferably a RPMI 1640-based culture medium comprising fetal bovine serum and penicillin-streptomycin solution. The final concentration of the fetal bovine serum is preferably 0.1 to 20%, more preferably 0.5 to 15%, and most preferably 1 to 10%. The final concentration of the penicillin-streptomycin solution is preferably 0 to 3%, more preferably 0.5 to 2% by volume.

In the present invention, the dead cell removal dye comprises an amine-reactive fluorescent dye that can permeate the envelope of damaged cells but cannot permeate the envelope of intact cells;

in the present invention, the solution obtained by dissolving the amine-reactive fluorescent dye with DMSO is preferably used as the dead cell removal dye, and the commonly used amine-reactive fluorescent dye includes Zombine Violet, alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, alexa Fluor700, APC/Cy7, APC/H7, brilliant Violet421, brilliant Violet510, brilliant Blue515, brilliant Violet 570, brilliant Violet 605, brilliant Violet 650, brilliant Violet711, brilliant Violet 785, FITC, LEAF, pacific Blue, PE/Cy5, PE/Cy7, PE/Dazzle, perCP, perCP/Cy5.5, and the amine-reactive fluorescent dye may be freely combined according to the laser and filter configuration of the flow cytometer, and the functions of the above described classes are the same.

In the present invention, the active ingredient of the FcR blocker solution comprises human immunoglobulins or unrelated immunoglobulins of the same genus and subtype as the streaming antibody used. The working solution of the FcR blocker solution has a mass concentration of preferably 0.1-100. Mu.g/mL, more preferably 1-10. Mu.g/mL.

In the present invention, the fluorescent species of the dead cell removal dye, fluorescent-labeled anti-human cell membrane surface molecule antibody include: alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, alexa Fluor700, APC/Cy7, APC/H7, brilliant Violet421, brilliant Violet510, brilliant Blue515, brilliant Violet 570, brilliant Violet 605, brilliant Violet 650, brilliant Violet711, brilliant Violet 785, FITC, LEAF, pacific Blue, PE/Cy5, PE/Cy7, PE/Dazzle, perCP, perCP/Cy5.5, and the like.

In the present invention, the fluorescent species may be freely combined according to the configuration of the laser and the filter of the flow cytometer.

In the present invention, the fluorescent-labeled antibody against a human cell membrane surface molecule comprises: anti-human CD3 epsilon antibodies, anti-human CD4 antibodies, anti-human CXCR5 antibodies, anti-human CCR6 antibodies, and anti-human CXCR3 antibodies; or anti-human CD3 epsilon antibodies, anti-human CD4 antibodies, anti-human CD8 antibodies, anti-human CXCR5 antibodies, anti-human CCR6 antibodies, and anti-human CXCR3 antibodies.

The invention also provides a method for detecting human CD3 by using the kit ⁺ CD4 ^- CD8 ⁺ A method of Tfh cells and subtypes thereof, comprising the steps of:

1) Mixing venous blood and blood diluent, placing the mixture on the upper layer of mononuclear cell separating liquid, setting the centrifugation condition to be 9, reducing the speed to be 0, 200-1000 g, and centrifuging for 15-35 min at 18-22 ℃, and sucking mononuclear cells, wherein the volume ratio of the venous blood, the blood diluent and the mononuclear cell separating liquid is 1: (0.5-2): (0.5-2);

2) Diluting the mononuclear cells obtained in the step 1) by using a blood diluent, setting the centrifugation condition to be 90-600 g, centrifuging for 4-30 min at 18-22 ℃, and collecting cell sediment;

3) Resuspending the cell pellet obtained in step 2) with a wash buffer to adjust the concentration of the cells to (1-10). Times.10 ⁶ Obtaining a first cell suspension by using the total cell mass per mL;

4) The first cell suspension obtained in the step 3) is subjected to a process of (0.1-1) x 10 ⁶ Mixing individual cells/100 mu L system with 0.1-10 mu L of DMSO dissolved dead cell removal dye and 0.1-10 mu L of FcR blocker solution, and incubating at 4-40 ℃ for 5-30 min to obtain first incubated cells;

5) The first incubated cells obtained in the step 4) are subjected to a procedure of (0.1-1). Times.10 ⁶ Mixing individual cells/100 mu L system with 0.5-5 mL cell staining buffer solution, centrifuging 90-600 g for 4-30 min, discarding supernatant, and precipitating cells according to (0.1-1) x 10 ⁶ Adding 0.5-20 mu L of fluorescent labeled antibody against human cell membrane surface molecules into the individual cells/100 mu L system, uniformly mixing, and then incubating for 5-30 min at 4-40 ℃ in a dark place to obtain second incubated cells;

6) The second incubated cells obtained in step 5) are subjected to a procedure of (0.1 to 1). Times.10 ⁶ Adding 0.5-5 mL of washing buffer solution into each cell/100 mu L system, uniformly mixing, centrifuging for 4-30 min at 90-600 g, discarding supernatant, and regulating the cell concentration to (0.1-10) x 10 by using the washing buffer solution ⁶ Detecting the expression of each antibody by a flow cytometer after each/mL; according to each antibodyExpression of body to calculate CD3 ⁺ CD4 ^- CD8 ⁺ Detection of Tfh subtype.

CD3 ⁺ CD4 ^- CD8 ⁺ Tfh(CD8 ⁺ Tfh) labeling method: CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ A cell;

CD3 ⁺ CD4 ^- CD8 ⁺ tfh subtype 1 (CD 8) ⁺ Labeling method of Tfh 1): CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ A cell;

CD3 ⁺ CD4 ^- CD8 ⁺ tfh subtype 2 (CD 8) ⁺ Labeling method of Tfh 2): CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ CCR6 ^- CXCR3 ^- Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ^- Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ^- A cell;

CD3 ⁺ CD4 ^- CD8 ⁺ tfh subtype 3 (CD 8) ⁺ Tfh 17) labeling method: CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells (-negative expression; + positive expression).

In the above method, it should be noted that:

the invention mixes venous blood with blood diluent, then places the mixture on the upper layer of mononuclear cell separating liquid, and sucks mononuclear cells after centrifugation for 15-35 min. The volume ratio of venous blood to blood diluent to mononuclear cell separating liquid in the invention is preferably 1: (0.5-2.0): (0.5 to 2.0), more preferably 1: (0.9-1.1): (0.9-1.1), most preferably 1:1:1; the centrifugal optimization of the invention is set to have an ascending speed of 9 and a descending speed of 0; the centrifugal force of the centrifugation is preferably 200 to 1000g, more preferably 250 to 950g, and most preferably 300 to 900g; the centrifugation time is preferably 15-25 min, and when the venous blood separation time is more than 2h, the centrifugation time is preferably 25-40 min; the temperature of the centrifugation is preferably 18-22 ℃; after centrifugation, the liquid is layered, the white membrane layer on the interface is carefully sucked out by a dropper, and the upper liquid is not sucked out as much as possible, so that the mononuclear cells are obtained.

After obtaining mononuclear cells, the invention dilutes the mononuclear cells with blood diluent and centrifugates for 4-30 min, and then collects cell sediment. The volume ratio of the mononuclear cells to the blood diluent in the invention is preferably 1: (0.5-50). The time of the centrifugation in the present invention is preferably 4 to 30 minutes, and the centrifugal force of the centrifugation is preferably 90 to 600g, more preferably 100 to 550g, and most preferably 150 to 500g.

After collecting the cell pellet, the present invention uses a washing buffer to re-suspend the cell pellet, and adjusts the concentration of the cells to (1-10) ×10 ⁶ And (3) obtaining a first cell suspension by per mL. In the present invention, the washing buffer is preferably added in two times, the first time for re-suspending the cell pellet, the cell count is performed after adding the peripheral blood mononuclear cells prepared from the whole blood with the volume of the washing buffer of 0.1-1.0 mL/mL, and then the washing buffer is added in a second time to make the final density of the cells be (1-10). Times.10 ⁶ And each mL.

The present invention provides a method of preparing the first cell suspension according to the formula (0.1-1) ×10 ⁶ After mixing the individual cells/100. Mu.L system with 0.1-10. Mu.L DMSO solution containing dead cell removal dye and 0.1-10. Mu.L FcR blocker solution, incubating for 5-30 min to obtain first incubated cells. In the present invention, the dead cell removal dye after lysis is preferably DMSO-solubilized. The temperature of the incubation is preferably 4-40 ℃, more preferably 15-35 ℃, the incubation is preferably performed in a light-proof environment, and the incubation time is preferably 5-30 min, more preferably 10-20 min.

After obtaining the first incubated cells, the invention subjects the first incubated cells to a procedure of (0.1-1). Times.10 ⁶ And uniformly mixing the individual cells/100 mu L system with 0.5-20 mu L of fluorescent labeled antibody against the surface molecules of the human cell membrane, and then incubating for 5-30 min at 4-40 ℃ in a dark place to obtain the second incubated cells. The temperature of the incubation is preferably 4-40 ℃, more preferably 15-35 ℃, the incubation is preferably performed in a light-proof environment, and the incubation time is preferably 5-30 min, more preferably 10-20 min.

The kit for detecting human blood follicular T helper cell subtype provided by the present invention will be described in detail with reference to examples.

Example 1

Kit for detecting subtype of human blood follicular T helper cells, wherein the blood follicular T helper cells are CD3 ⁺ CD4 ^- CD8 ⁺ Tfh cells;

Wherein, the blood diluent and the washing buffer solution are solutions with the osmotic pressure equal to that of human plasma,

specifically, the blood diluent and wash buffer are PBS buffers.

The PBS buffer solution comprises the following components in parts by weight: 8.0 parts of NaCl,0.20 parts of KCl and 1.16 parts of Na ₂ HPO ₄ And 0.20 part KH ₂ PO ₄ PBS buffer pH 7.4

The source of the blood diluent is PBS buffer, which is self-formulated or commercially available from the above components.

The mononuclear cell separation liquid (Serumwerk Bernburg AG, product number 1858) contains polysucrose and diatrizamine as effective components, the mononuclear cell separation liquid is distilled water as solvent, the mononuclear cell separation liquid has a density of 1.077g/mL and an osmotic pressure of 290mOsm.

The cell culture solution is a culture solution for culturing human blood cells.

The cell culture medium was based on RPMI 1640 and contained a solution of fetal bovine serum and penicillin-streptomycin (soribao, cat# P1400) at a final concentration of 5% by volume and penicillin-streptomycin solution at a final concentration of 1% by volume.

The RPMI 1640 basal medium was an aqueous solution containing the following components (see Table 1):

TABLE 1 composition of basal medium

The dead cell removal dye is an amine reactive fluorescent dye which can penetrate through the cell membrane of the damaged cell but cannot penetrate through the cell membrane of the whole cell; dead cell removal dye was a solution obtained by dissolving amine-activated fluorescent dye (Biolegend, cat. 423114) with anhydrous DMSO (Thermo filter, D12345).

The active ingredient of the FcR blocker (Biolegend, cat. No. 422302) solution is human immunoglobulin, and the mass concentration of the FcR blocker solution is 5 μg/mL.

The types of dead cell removal dye and fluorescent dye of the fluorescent labeled anti-human cell membrane surface molecular antibody are Zombie Viole, perCP/Cy5.5-CD3 epsilon, FITC-CD4, APC/Cy7-CXCR5, PE-CCR6 and Brilliant Viole 510-CXCR3.

Example 2

specifically, the blood diluent and wash buffer are PBS buffers.

The PBS buffer solution comprises the following components in parts by weight: 8.0 parts of NaCl,0.20 parts of KCl and 1.16 parts of Na ₂ HPO ₄ And 0.20 part KH ₂ PO ₄ The pH of the PBS buffer was 7.3.

The mononuclear cell separation liquid (Serumwerk Bernburg AG, product number 1858) contains diatrizoic sodium and polysaccharide as effective components, distilled water as solvent, and has density of 1.077g/mL and osmotic pressure of 290mOsm.

The cell culture medium was based on RPMI 1640 and contained fetal bovine serum and penicillin-streptomycin solution, the final concentration of the fetal bovine serum was 7% by volume and the final concentration of penicillin-streptomycin solution (soribao, cat# P1400) was 1.5% by volume.

The RPMI 1640 basal medium was an aqueous solution containing the components shown in Table 1 (see Table 1).

The dead cell removal dye is an amine reactive fluorescent dye which can penetrate through the cell membrane of the damaged cell but cannot penetrate through the cell membrane of the whole cell; dead cell removal dye was a solution obtained by dissolving amine-activated fluorescent dye (Biolegend, cat. 423106) with anhydrous DMSO (Thermo filter, D12345).

The active ingredient of the FcR blocker (Biolegend, cat. No. 422302) solution is an irrelevant immunoglobulin of the same species and subtype as the used flow antibody, and the mass concentration of the FcR blocker solution working solution is 50 μg/mL.

The types of dead cell removal dye and fluorescent dye of the fluorescent labeled anti-human cell membrane surface molecular antibody are Alexa Fluor 488, zombie NIR, perCP/Cy5.5-CD3 epsilon, FITC-CD4, APC-CXCR5, PE-CCR6 and Brilliant Violet510-CXCR3.

Example 3

specifically, the blood dilution and wash buffer are PBS buffers.

The active ingredient of the mononuclear cell separation liquid (Serumwerk Bernburg AG, product number 1858) is iodixanol, the solvent of the mononuclear cell separation liquid is distilled water, the density of the mononuclear cell separation liquid is 1.077g/mL, and the osmotic pressure is 290mOsm.

The cell culture solution is based on RPMI 1640 and comprises fetal bovine serum and penicillin-streptomycin solution, wherein the final volume concentration of the fetal bovine serum is 10%, and the final volume concentration of the penicillin-streptomycin solution is 1%.

The RPMI 1640 basal medium was an aqueous solution of the components shown in Table 1.

The dead cell removal dye is an amine reactive fluorescent dye which can penetrate through the cell membrane of the damaged cell but cannot penetrate through the cell membrane of the whole cell; dead cell removal dye was a solution obtained by dissolving amine-activated fluorescent dye (Biolegend, zombie Aqua) with anhydrous DMSO (Thermo filter, D12345).

The active ingredient of the FcR blocker (Biolegend, cat. No. 422302) solution is human immunoglobulin, and the mass concentration of the FcR blocker solution working solution is 5 mug/mL.

The types of dead cell removal dye and fluorescent dye of the fluorescent labeled anti-human cell membrane surface molecular antibody are PerCP/Cy5.5-CD3 epsilon, FITC-CD4, APC-CXCR5, PE-CCR6 and Brilliant Violet421-CXCR3.

Example 4

Wherein the blood diluent and the washing buffer solution are solutions with the osmotic pressure equal to that of human plasma, and concretely, the blood diluent and the washing buffer solution are NaCl solutions, and the mass concentration of the NaCl solutions is 0.9%.

The dead cell removal dye is an amine reactive fluorescent dye which can penetrate through the cell membrane of the damaged cell but cannot penetrate through the cell membrane of the whole cell; dead cell removal dye was a solution obtained by dissolving amine-activated fluorescent dye (Biolegend, cat. 423112) with anhydrous DMSO (Thermo filter, D12345).

The types of dead cell removal dye and fluorescent dye of the fluorescent labeled anti-human cell membrane surface molecular antibody are PerCP/Cy5.5-CD3 epsilon, APC/Cy7-CD4, APC-CXCR5, PE-CCR6 and Brilliant Violet510-CXCR3.

The kit provided by the embodiments 1 to 4 of the invention has simple preparation method and simple and convenient operation, and can be used for detecting human CD3 ⁺ CD4 ^- CD8 ⁺ Tfh cells and subtypes thereof. The detection of human CD3 using this kit will be described below by taking example 1 as an example ⁺ CD4 ^- CD8 ⁺ Tfh cells and subtypes thereof.

Example 5

Human CD3 detection kit for detecting human blood follicular T helper cell subtype ⁺ CD4 ^- CD8 ⁺ The method for detecting Tfh cells and subtype thereof is shown in FIG. 1, and specifically comprises the following steps:

s1, mixing venous blood with blood diluent, placing the mixture on the upper layer of mononuclear cell separating liquid, setting the centrifugation condition to be 9, reducing the speed to be 0, taking 600g of cell sediment on the lower layer, centrifuging at 20 ℃ for 25min, and sucking mononuclear cells, wherein the volume ratio of the venous blood, the blood diluent and the mononuclear cell separating liquid is 1:1:1, obtaining mononuclear cells after centrifugation;

s2, diluting the mononuclear cells obtained in the step S1 by using blood diluent, setting the centrifugation condition to be 325g at 20 ℃, and collecting cell sediment after 20min of centrifugation;

s3, re-suspending the cell pellet obtained in S2 with a washing buffer to adjust the concentration of the cells to 5X 10 ⁶ Obtaining a first cell suspension by using the total cell mass per mL;

s4, mixing the first cell suspension obtained in S3 according to 0.5X10 ⁶ Individual cells/100. Mu.L System 5.5. Mu.L of LDMSO lysed dead cell removal dye and 5.5. Mu.LAfter mixing the FcR blocker solution, incubating for 15min at 25 ℃ to obtain first incubated cells;

s5, first incubation cells obtained in S4 were incubated according to 0.5X10 ⁶ The individual cells/100. Mu.L system was mixed with 2.3mL of cell staining buffer, centrifuged at 345g for 17min, and the supernatant was discarded and the cell pellet was then purified according to 5.5X10 ⁶ Adding 10 mu L of fluorescent labeled antibody against human cell membrane surface molecules into the individual cells/100 mu L system, uniformly mixing, and incubating for 15min at 25 ℃ in a dark place to obtain second incubated cells;

s6, incubating the second incubated cells obtained in S5 according to 5.5X10 ⁶ Adding 3.25mL of washing buffer solution into each cell/100 mu L system, uniformly mixing, centrifuging for 17min at 345g, discarding the supernatant, and adjusting the cell concentration to 5.5X10 by using the washing buffer solution ⁶ Detecting the expression of each antibody by a flow cytometer after each/mL; calculation of CD3 based on the expression of each antibody ⁺ CD4 ^- CD8 ⁺ Detection of Tfh subtype.

CD3 ⁺ CD4 ^- CD8 ⁺ Tfh(CD8 ⁺ Tfh) includes: CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ Cells (-negative expression; + positive expression);

CD3 ⁺ CD4 ^- CD8 ⁺ tfh subtype 1 (CD 8) ⁺ Tfh 1) includes: CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ CCR6 ^- CX CR3 ⁺ Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ Cells (-negative expression; + positive expression);

CD3 ⁺ CD4 ^- CD8 ⁺ tfh subtype 2 (CD 8) ⁺ Tfh 2) includes: CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ CCR6 ^- CX CR3 ^- Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ^- Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ^- Cells (-negative expression; + positive expression);

CD3 ⁺ CD4 ^- CD8 ⁺ tfh subtype 3 (CD 8) ⁺ Tfh 17) includes: CD3 epsilon ⁺ CD4 ^- CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR 5 ⁺ CCR6 ⁺ CXCR3 ^- Cells (-negative expression; + positive expression).

In S1, when the venous blood separation time is more than 2 hours, the centrifugation time is 25-40 min.

In S2, the volume ratio of mononuclear cells to blood diluent is preferably 1: (0.5-50).

In S3, the washing buffer is added twice, the first time is used for re-suspending cell precipitation, the cell count is carried out after the peripheral blood mononuclear cells prepared by whole blood with the volume of 0.1-1.0 mL/mL are added into the washing buffer, and then the washing buffer is added for the second time, so that the final density of the cells is (1-10) multiplied by 1 ⁶ And each mL.

The invention mixes venous blood with blood diluent, then places the mixture on the upper layer of mononuclear cell separating liquid, and sucks mononuclear cells after centrifugation for 15-35 min.

In S4, the incubation is performed in a dark environment.

The invention provides a method for detecting human CD3 ⁺ CD4 ^- CD8 ⁺ A kit of Tfh cells and subtypes thereof, and a detection method thereof. The kit has the advantages of simple and easily obtained components, and a rapid and convenient detection method, and can be used for auxiliary diagnosis of sepsis.

The method is characterized in that the blood samples of sepsis patients of the severe medical department of Henan province people hospital, allergic asthma patients of the allergic reaction department and healthy people of the physical examination center are selected for detecting the CD3 of the human by the flow cytometry ⁺ CD4 ^- CD8 ⁺ The kit of Tfh cells and their subtype is described in detail.

The specific detection method comprises the following steps:

1) Peripheral venous blood was collected from healthy (n=14), allergic asthma (n=18) and sepsis (n=13);

2) Taking 1mL of fresh peripheral venous blood (blood in vitro is less than or equal to 2 h), and adding 1mL of blood diluent to dilute the blood;

3) Taking 1mL of mononuclear cell separating liquid at the bottom of a centrifuge tube, carefully superposing diluted blood on the mononuclear cell separating liquid, and avoiding mixing the blood and the mononuclear cell separating liquid;

4) Putting the mixture into a centrifugal machine, centrifuging for 20min at 800g, setting the temperature of the centrifugal machine to 20 ℃, setting the rising speed to 9 and setting the falling speed to 0;

5) Sucking out the tunica media (mononuclear cells) on the interface by using a dropper, and avoiding sucking out the upper liquid as much as possible;

6) 2mL of blood diluent is used for diluting mononuclear cells, and 250g of the mononuclear cells are centrifuged for 10min at room temperature; cell pellet was collected, and cell count was performed after resuspension of mononuclear cells with 100. Mu.L of cell culture medium, and cell density was adjusted to 4X 10 with cell culture medium ⁶ The number of mononuclear cells obtained per mL is shown in FIG. 2;

7) 100. Mu.L of cell suspension was added per tube, and then 1. Mu.L of DMSO-dissolved dead cell removal dye Zombie Violet and 5. Mu.L of FcR blocker solution were added and incubated at room temperature for 15min in the absence of light;

8) mu.L of PerCP/Cy5.5 labeled anti-human CD3 epsilon antibody, 5 mu.L of FITC labeled anti-human CD4 antibody, 5 mu.L of APC/Cy7 labeled anti-human CXCR5 antibody, 5 mu.L of PE labeled anti-human CCR6 antibody, 5 mu. L Brilliant Violet510 (BV 510) labeled anti-human CXCR3 antibody were added per tube and incubated at room temperature for 15min in the absence of light;

9) Centrifuging at room temperature of 200g for 6min, and discarding supernatant;

10 1.5mL of washing buffer solution is added into each tube, 200g of washing buffer solution is centrifuged for 6min at room temperature, and the supernatant is discarded;

11 200 μl wash buffer per tube was added to resuspend cells and flow cytometry was used to detect expression levels of CD3 epsilon, CD4, CXCR5, CCR6 and CXCR 3;

12 The expression level of each molecule in blood of healthy people, allergic asthma patients and sepsis patients is shown in figures 3-4;

CD3ε ⁺ CD4 ^- CXCR5 ⁺ cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXC R5 ⁺ The cells belong to CD3 ⁺ CD4 ^- CD8 ⁺ Tfh(CD8 ⁺ Tfh)；

CD3ε ⁺ CD4 ^- CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ⁺ The cells belong to CD3 ⁺ CD4 ^- CD8 ⁺ Tfh subtype 1 (CD 8) ⁺ Tfh1)；

CD3ε ⁺ CD4 ^- CXCR5 ⁺ CCR6 ^- CXCR3 ^- Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ^- Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ^- CXCR3 ^- The cells belong to CD3 ⁺ CD4 ^- CD8 ⁺ Tfh subtype 2 (CD 8) ⁺ Tfh2)；

CD3ε ⁺ CD4 ^- CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells or CD3 epsilon ⁺ CD8 ⁺ CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- Cells or CD3 epsilon ⁺ CD4 ^- CD8 ⁺ CXCR5 ⁺ CCR6 ⁺ CXCR3 ^- The cells belong to CD3 ⁺ CD4 ^- CD8 ⁺ Tfh subtype 3 (CD 8) ⁺ Tfh17)；

CD3 epsilon in peripheral venous mononuclear cell populations of healthy people, allergic asthma patients and sepsis patients ⁺ CD 4 ^- T cells, CD8 ⁺ Tfh cells, CD8 ⁺ Tfh1 cells, CD8 ⁺ Tfh2 cells, CD8 ⁺ The expression levels of Tfh17 cells are shown in figure 5.

As can be seen from the above examples, the kit and the detection method thereof of the present invention can rapidly detect CD3 in human peripheral blood ⁺ CD4 ^- CD8 ⁺ Tfh cells and subtypes thereof, andis used for assisting in diagnosis of allergic asthma and sepsis.

It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A kit for detecting human blood follicular T helper cell subtype is characterized in that the blood follicular T helper cell is CD3 ⁺ CD4 ^- CD8 ⁺ Tfh cells, the kit comprises blood dilutions, mononuclear cell separation, cell culture, dead cell removal dye, fcR blocker solution, fluorescently labeled antibodies against human cell membrane surface molecules, and wash buffer.

2. A kit for detecting a human blood follicular T helper cell subtype according to claim 1,

the blood dilution comprises NaCl solution or PBS buffer solution; the mass concentration of the NaCl solution is 0.85-0.95%;

The penetration concentration of the PBS is 280-320 mMol/L;

the pH value of the PBS buffer solution is 7.0-7.6.

3. The kit for detecting human blood follicular T helper cell subtype according to claim 1, wherein the mononuclear cell separation solution has a density of 1.076 to 1.078g/mL and an osmotic pressure of 275 to 305mOsm.

4. The kit for detecting human blood follicular T helper cell subtype according to claim 1, wherein the cell culture fluid is formulated as follows: RPMI 1640 is used as a basic culture medium, and fetal bovine serum and penicillin-streptomycin solution are added into the RPMI 1640; the final concentration of the fetal bovine serum is 0.5-15%; the final concentration of the penicillin-streptomycin solution is 0.5-2%.

5. A kit for detecting a human blood follicular T helper cell subtype according to claim 1, wherein the dead cell removal dye is an amine reactive fluorescent dye.

6. A kit for detecting a subtype of human blood follicular T helper cells according to claim 1, wherein the FcR blocker solution comprises human immunoglobulins or unrelated immunoglobulins of the same genus and subtype as the streaming antibody used.

7. A kit for the surgical detection of human blood follicular T helper cell subtype according to claim 1, wherein the fluorescent species of the dead cell removal dye and fluorescently labeled antibody against human cell membrane surface molecules comprises fluorescence of one or more of Alexa Fluor 488, alexa Fluor 594, alexa Fluor 647, alexa Fluor700, APC/Cy7, APC/H7, brilian Violet421, brilian Violet510, brilian Blue515, brilian Violet 570, brilian Violet 605, brilian Violet 650, brilian Violet711, brilian Violet 785, FITC, LEAF, pacific Blue, PE/Cy5, PE/Cy7, PE/Dazzle, perCP or PerCP/5.5.

8. The kit for detecting a human blood follicular T helper cell subtype according to claim 7, wherein said fluorescently labeled antibodies against human cell membrane surface molecules comprise anti-human CD3 epsilon antibodies, anti-human CD4 antibodies, anti-human CXCR5 antibodies, anti-human CCR6 antibodies and anti-human CXCR3 antibodies; or anti-human CD3 epsilon antibodies, anti-human CD4 antibodies, anti-human CD8 antibodies, anti-human CXCR5 antibodies, anti-human CCR6 antibodies, and anti-human CXCR3 antibodies.

9. Use of a kit for detecting human blood follicular T helper cell subtype according to claims 1 to 8 for the diagnosis of allergic diseases and sepsis.

10. A method for detecting human blood follicular T helper cell subtype using the kit for detecting human blood follicular T helper cell subtype according to claims 1 to 8, comprising the steps of:

s1, separating mononuclear cells from peripheral venous blood of a human body;

s5, adding the second incubated cells obtained in the step S4 into a washing buffer, uniformly mixing, centrifuging, discarding the supernatant, and washingThe cell concentration was adjusted to 10 with buffer ⁵ ～10 ⁶ Detecting the expression of each antibody by a flow cytometer after each cell/mL; calculation of CD3 based on the expression of each antibody ⁺ CD4 ^- CD8 ⁺ Detection of Tfh cells and subtypes thereof.