A bovine peripheral blood CD4 + CD8 + T lymphocyte flow cytometry kit (indirect fluorescence method)

Abstract

The present application relates to a kind of peripheral blood CD4+, CD8+ T lymphocyte flow cytometry detection kit (indirect fluorescence method) of cow.In the lymphocyte population defined, CD3, CD4 All are positive or CD3, CD8 All are positive in subpopulation of cell, and the cell number or proportion of double-positive cell is calculated.

Description

Technical Field

[0001] This invention relates to the field of veterinary medicine, specifically to a bovine peripheral blood CD4... + CD8 + T lymphocyte flow cytometry kit (indirect fluorescence method). Background Technology

[0002] CD3 molecules are important surface markers of T cells. + Lymphocytes represent all T lymphocytes. CD4 + T lymphocytes are CD3 and CD4 positive cells, and also CD8 positive cells. + T lymphocytes are CD3 and CD8 positive cells. Studies suggest that the ratio of lymphocyte subsets is an important indicator of a dairy cow's immune capacity, and that CD4+ levels in dairy cow blood... + / CD8 + The T lymphocyte ratio was significantly negatively correlated with somatic cell count in milk, which is of great significance for the diagnosis of mastitis in dairy cows; CD8 + The proportion of T lymphocytes is significantly correlated with fertility and can serve as a useful marker for improving the reproductive performance of dairy cows.

[0003] Currently detecting CD4 + T, CD8 + T lymphocytes are mainly detected by flow cytometry and magnetic bead sorting.

[0004] Flow cytometry uses monoclonal antibodies to quantitatively analyze specific cell types present in a sample, enabling high-throughput screening of multiple parameters within a single sample. Bovine peripheral blood CD4 or CD8 cells do not fluoresce naturally. Monoclonal antibodies labeled with fluorescent dyes bind to specific antigens on the cell surface. When the cells pass through the laser beam of a flow cytometer, a fluorescent signal appears, and the cell count is calculated by measuring the intensity of the fluorescence signal. In flow cytometry analysis of human (or mouse) T lymphocytes, CD4 cells are identified within the cell population identified by CD3 fluorescent antibodies. + CD8 + T lymphocyte subsets. In cattle, positive cells detected by flow cytometry using only CD4 or CD8-specific monoclonal fluorescent antibodies include not only the target cells but also other cell types of non-T lymphocytes, such as CD3-negative CD3+ cells. - CD4 + Cells or CD3 - CD8 + The accuracy is not high.

[0005] The Magnetic Bead Sorting (MACS) method labels one of the two specific F(ab) segments of a particular monoclonal antibody's immunoglobulin with a magnetic bead, while the other bead specifically binds to the antigenic determinant of a cell surface antigen. This allows the labeled CD4+ to bind to the target cell surface antigen. + CD8 + After passing through MACS, T lymphocytes are sorted by magnetic adsorption. MACS can only sort based on one parameter at a time, and its purity is lower than that of flow cytometry. This method is also used for sorting CD3+ cells. + CD4 + CD3 + CD8 + Identifying double-positive T cells is not feasible.

[0006] The reasons for these problems and drawbacks: Choosing different fluorescent dyes is often used to identify different cell subpopulations, while simultaneously detecting CD3. + CD4 + CD3 + CD8 + For double-positive cells, different fluorescent labels for CD3, CD4, and CD8 antibodies are required, and these labels must correspond to different receiver channels on the flow cytometer. Currently, there is a limited number of commercially available fluorescently labeled bovine CD4 and CD8 antibodies, and even fewer CD3-labeled antibodies, such as BIO-RAD's Mouse anti-Bovine CD3 (MCA6080) and Invitrogen's MA5-28343. All of these antibodies use the MM1A antigen number and do not carry a fluorescent label, requiring the selection of a suitable fluorescently labeled secondary antibody for flow cytometry analysis. While there are many CD3 antibodies available for humans and mice, the expression of T cell surface antigens varies significantly between different species, resulting in low cross-reactivity. Therefore, fluorescently labeled anti-human (or mouse) CD3 antibodies cannot be used for bovine CD3. + T lymphocyte detection, such as using Bio-Rad's RAT ANTI HUMAN CD3 (MCA1477) to detect CD3 in bovine peripheral blood, showed no detection. + Positive. In addition, the simultaneous comprehensive sorting of multiple parameters including CD3, CD4 and CD8 places higher demands on pretreatment conditions such as antibody dosage and incubation time. Summary of the Invention

[0007] The purpose of this invention is to provide bovine peripheral blood CD4 + CD8 + A flow cytometry method for T lymphocyte detection. This method addresses the challenge of detecting bovine CD3-positive cells using flow cytometry. It involves selecting appropriate fluorescently labeled secondary antibodies based on the species origin and antibody subtype of the primary antibody, and optimizing pretreatment and detection conditions before flow cytometry to ensure a high detection rate of CD3-positive cells.

[0008] On the other hand, solving CD3 + CD4 + and CD3 + CD8 + The detection of double-positive cells involves screening for cell subsets that are positive for both CD3 and CD4 or both CD3 and CD8 from a defined T lymphocyte population, and then calculating the content of double-positive cells.

[0009] The solution of this invention is as follows: a bovine peripheral blood CD4 + CD8 + The T lymphocyte flow cytometry assay kit (indirect fluorescence method) includes the following steps:

[0010] (1) Use EDTA anticoagulant blood collection tubes to collect whole blood from the cattle to be tested. Set up sample tubes, blank control tubes, and single positive control tubes. The single positive control tubes include CD3 single positive tubes, CD4 single positive tubes, and CD8 single positive tubes, also known as single staining tubes and isotype control tubes. The isotype control tubes include CD4 isotype control tubes and CD8 isotype control tubes.

[0011] (2) After adding the corresponding antibody to the sample tube and the control tube, mix well and incubate at 4°C in the dark for 30 min. Mix well again every 5 min during the incubation process.

[0012] (3) After incubation and staining, add red blood cell lysis buffer to lyse the cells, then centrifuge to discard the supernatant, disperse the cells, wash the cells, resuspend the cells, and prepare blank control tubes, single positive control tubes (except CD3 single positive tubes) and isotype control tubes for instrument detection.

[0013] (4) Add 30 μl of 1:100 fluorescent secondary antibody to the sample tube and CD3 single positive tube, and incubate at 4°C in the dark for 30 min. The fluorescent secondary antibody is prepared by dissolving 10 μl of Goat anti Mouse IgG1:FITC (STAR132F) in 990 μl of staining buffer.

[0014] (5) After incubation, the cells are centrifuged again to discard the supernatant, dispersed, washed, and resuspended. The CD3 single-positive tubes are ready for instrumental testing.

[0015] (6) Add 5 μl of Mouse anti Bovine CD4:Alexa Fluor 647 and 5 μl of Mouse anti Bovine CD8:RPE to the sample tube, incubate at 4°C in the dark for 0.5 h, and then perform instrument detection.

[0016] Furthermore, the antibody added to the sample tube in the second step is Mouse anti-Bovine CD3, with an addition volume of 2.5 μl.

[0017] Furthermore, the blank control tube, single positive control tube, and isotype control tube are mixed blood samples from all the blood samples to be tested.

[0018] Furthermore, the control tube includes a blank control tube, a CD3 single-positive tube, a CD4 single-positive tube, a CD8 single-positive tube, a CD4 isotype control, and a CD8 isotype control. The antibodies added in the second step are: staining buffer, Mouse anti-Bovine CD3, Mouse anti-Bovine CD4: Alexa Fluor 647, Mouse anti-Bovine CD8: RPE, Mouse IgG2a Negative Control: Alexa Fluor, and Mouse IgG2a Negative Control: RPE.

[0019] Furthermore, the specific operation of centrifugation and supernatant disposal is as follows: centrifuge at 4℃ and 500g for 5 minutes, discard the supernatant after centrifugation, and disperse the cells.

[0020] Furthermore, the specific procedure for washing the cells is as follows: wash once with 1 ml of Flow Cytometry Staining Buffer, centrifuge at 500g for 5 min at 4°C, discard the supernatant, and disperse the cells.

[0021] Furthermore, the specific operation of resuspending cells is as follows: add 300 μl of Flow Cytometry Staining Buffer to resuspend cells.

[0022] Furthermore, the amounts of antibody placed in the blank control tube, CD3 single positive tube, CD4 single positive tube, CD8 single positive tube, CD4 isotype control tube, and CD8 isotype control tube in the second step are 5 μl, 2.5 μl, 5 μl, 5 μl, 5 μl, and 5 μl, respectively.

[0023] When testing CD3, CD4, and CD8 single-positive tubes, this invention sets up different concentration gradients of antibodies. For the recommended 100μl anticoagulation test, the optimal dosages of the three antibodies, Mouse anti-Bovine CD3, Mouse anti-Bovine CD4:AlexaFluor 647, and Mouse anti-Bovine CD8:RPE, are 2.5μl, 5μl, and 5μl, respectively.

[0024] When detecting CD3 single-positive cells, when adding fluorescein-labeled secondary antibody (Goat anti Mouse IgG1:FITC) to the cell suspension after incubation with primary antibody (Mouse anti Bovine CD3), different concentration gradients should be set. It is recommended to first dilute the fluorescent secondary antibody 1:100, and then add 30 μl of the fluorescent secondary antibody dilution to 300 μl of the suspended cell solution.

[0025] The beneficial effects of this invention are:

[0026] Based on the antibody subtype (IgG1) and species origin (mouse) of the primary antibody (Mouse anti-Bovine CD3), a corresponding anti-mouse IgG1 fluorescent secondary antibody was selected. Antibody dilution and dosage were optimized to assemble a bovine peripheral blood CD4 antibody. + CD8 + T lymphocyte flow cytometry assay kit (indirect fluorescence method). CD4 monoclonal antibody and CD8 monoclonal antibody labeled with fluorescent dyes corresponding to different detection channels are selected to detect CD3. + CD4 + and CD3 + CD8 + Double-positive cells.

[0027] CD4 + :CD8 + The ratio is a cell-mediated adaptive immune-related trait that is correlated with udder health, reproductive performance, and production performance in dairy cows. This invention establishes a method for detecting bovine peripheral blood CD4 using indirect fluorescence flow cytometry. + CD8 + The T lymphocyte method is beneficial for assessing the health status of cattle, and the accumulation of large amounts of data can be used to study bovine CD3... + CD4 + CD3 + CD8 + or CD4 + :CD8 + Genetic selection of traits may become a useful tool for improving animal health, adaptability, and fertility.

[0028] This invention includes a blank control, using the same anticoagulated blood as the test sample, except that a staining buffer is used instead of a fluorescent antibody; all other treatments are identical. The blank control is generally used as a reference for voltage adjustment. When adjusting the voltage on the instrument, a blank tube is first run to adjust the voltage parameters so that the cell signal appears at the appropriate position in the FSC and SSC channels. Simultaneously, the background signal of the cells can be observed, ensuring that the blank tube does not show the expected signal of the stained sample, or is excluded directly during the gate selection.

[0029] A single-positive control was set up for single-stain adjustment and compensation, which means subtracting the signal that leaked out in other detection channels for that fluorescence. Multicolor flow cytometry is prone to fluorescence leakage. The single-stain control can show the level of spectral overlap between different fluorophores and thereby remove or compensate for the overlap between them.

[0030] An isotype control was included to eliminate background staining caused by non-specific binding of antibodies to Fc receptors on the cell surface and electrostatic binding. This accurately eliminates false positives and helps verify antibody specificity. Antibodies of the same species, subtype, subchain, and fluorescent label, purified from the serum of unimmunized animals, are generally selected.

[0031] The above-mentioned blank control, single-positive control, and isotype control settings ensure the specificity of the detection results. This invention first detects CD3 to delineate total T lymphocytes, and then uses CD4 antibody and CD8 antibody to distinguish CD4 cells. + and CD8 + T lymphocytes, compared with detection using only CD4 or CD8 antibodies, showed an increase in CD3. + CD4 + or CD3 + CD8 + The accuracy of double-positive cells. During the test, 10 different blood samples were selected for two repeated tests, CD3. + CD4 + The coefficient of variation for repeated assays of double-positive T cells was 2.41%, CD3 + CD8 + The coefficient of variation for double-positive T cells was 9.02%. In summary, this kit demonstrates good specificity, accuracy, and reproducibility, and shows promising application prospects in bovine immune function analysis and the selection of healthy cattle. Attached Figure Description

[0032] Figure 1 The image shows the scattered light signal used to distinguish lysed bovine peripheral blood cell populations.

[0033] Figure 2 The image shows the cell distribution in each fluorescence channel of the CD3 single-stain tube;

[0034] Figure 3 The image shows the cell distribution in each fluorescence channel of a CD4 single-staining tube;

[0035] Figure 4 The image shows the cell distribution in each fluorescence channel of a CD8 single-stain tube;

[0036] Figure 5 The image shows the cell distribution in the CD4 isotype control tube under the APC fluorescence channel.

[0037] Figure 6 The image shows the cell distribution in the CD8 isotype control tube under the PE fluorescence channel;

[0038] Figure 7 The image shows flow cytometry analysis of CD3 / CD4 and CD3 / CD8 double-positive cells in a peripheral blood sample.

[0039] Figure 8 The image shows flow cytometry analysis of 2CD3 / CD4 and CD3 / CD8 double-positive cells in a peripheral blood sample.

[0040] Figure 9 The image shows flow cytometry analysis of 3CD3 / CD4 and CD3 / CD8 double-positive cells in a peripheral blood sample. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments:

[0042] The flow cytometer used in this invention is a Cytoflex S model, sourced from Beckman Coulter.

[0043] Example 1

[0044] 1. Preparation of experimental materials

[0045] Consumables: EDTA blood collection tubes, ice packs, foam box, EP tubes (1.5mL), red blood cell lysis buffer, FlowCytometry Staining Buffer, three pipettes (1ml, 20-200μl, 0.5-20μl), two boxes of pipette tips, centrifuge, flow cytometry tubes, 200-mesh filter.

[0046] Antibody reagents: ① Mouse anti-Bovine CD3, ② Mouse anti-Bovine CD4: Alexa Fluor 647, ③ Mouse

[0047] anti Bovine CD8:RPE, ④Mouse IgG2a Negative Control:Alexa Fluor, ⑤Mouse IgG2a Negative Control:RPE, ⑥Goat anti Mouse IgG1:FITC

[0048] 2. Reagent Preparation

[0049] 1:100 secondary antibody: 10 μl Goat anti Mouse IgG1:FITC (STAR132F) dissolved in 990 μl staining buffer

[0050] Flow Cytometry Staining Buffer: 1×Calcium- and Magnesium-Free PBS + 1% BSA + 2mM EDTA + 0.1% Sodium Azide (NaN3)

[0051] FlowClean solution: Dilute 2% sodium hypochlorite solution 5 to 6 times and filter with a 0.2 μm filter membrane.

[0052] 3-stream pre-processing

[0053] (1) Use 5ml EDTA anticoagulant blood collection tubes to collect whole blood from 3 cows as 3 blood samples to be tested. Shake well to ensure that the anticoagulant is fully combined with the blood, place in an ice box and transport back to the laboratory.

[0054] (2) Take 400 μl of each of the three blood samples collected in the previous step and mix them evenly in a 1.5 ml EP tube (any blood sample can be selected if there are many samples). Then take 100 μl of anticoagulated blood from the above EP tubes and mix them into 6 EP tubes, and label them as Blank (blank control), CD3 single staining tube, CD4 single staining tube, CD8 single staining tube, CD4 isotype control, and CD8 isotype control, respectively.

[0055] (3) Take 100 μl of each blood sample to be tested into 3 different EP tubes and label them as sample 1, 2 and 3 respectively.

[0056] (4) Add the corresponding antibodies to each tube produced in steps 2 and 3 and mix well. The types and amounts of antibodies added are shown in Table 1. It is recommended to use the tube-flicking method to mix well, and try not to blow with the pipette tip. After mixing well, incubate at 4°C in the dark for 30 minutes. During this process, the cells will bind to the antibodies. Mix well every 5 minutes during the incubation process to make the binding more complete.

[0057] Table 1 shows the antibody names and dosages added to each tube.

[0058] Blank Staining buffer 5μl CD3 Single Color Tube Mouse anti-Bovine CD3 2.5μl CD4 Single-color tube Mouse anti Bovine CD4:Alexa Fluor 647 5μl CD8 Single-color tube Mouse anti-Bovine CD8:RPE 5μl CD4 isotype comparison Mouse IgG2a Negative Control:Alexa Fluor 5μl CD8 isotype control Mouse IgG2a Negative Control:RPE 5μl Samples 1, 2, and 3 Mouse anti-Bovine CD3 2.5μl

[0059] (5) Add 1 ml of red blood cell lysis buffer to each tube and lyse on ice for 5 min. During the lysis process, mix the cells thoroughly to ensure complete dissolution and more complete lysis.

[0060] (6) Centrifuge each tube after lysis at 4℃ and 500g for 5 minutes. After centrifugation, discard the supernatant and disperse the cells.

[0061] (7) Wash the cells. Add 1 ml of Flow Cytometry Staining Buffer to each tube and wash once. Centrifuge at 500 g for 5 min at 4℃, discard the supernatant, and disperse the cells.

[0062] (8) Then add 300 μl of Flow Cytometry Staining Buffer to each tube to resuspend the cells.

[0063] (9) Store Blank, CD4 monostained tubes, CD8 monostained tubes, CD4 isotype control, and CD8 isotype control tubes at 4°C in the dark until they are ready for instrument testing.

[0064] (10) Add 30 μl of 1:100 fluorescent secondary antibody to CD3 single staining tubes and sample tubes 1, 2 and 3 respectively, and incubate at 4℃ in the dark for 30 min.

[0065] (11) Centrifuge the four tubes from the previous incubation at 4°C and 500g for 5 minutes, discard the supernatant, and disperse the cells. Then proceed according to (7).

[0066] (8) Step: Clean the cells.

[0067] (12) Store CD3 single-color tubes at 4°C away from light until they are ready for testing.

[0068] (13) Add 5 μl Mouse anti Bovine CD4:Alexa Fluor 647 and 5 μl Mouse anti Bovine CD8:RPE to sample tubes 1, 2 and 3, and incubate at 4°C in the dark for 30 min.

[0069] (14) During sample processing, check the sheath fluid tank to confirm that there is sufficient sheath fluid and that it does not exceed the upper limit indicated. At the same time, empty the waste fluid tank, turn on the instrument power, open the CytExpert software, and ensure that the online operation is normal.

[0070] (15) Add 2 mL of FlowClean solution to an unused sample tube, insert the tube into the sample holder, and select run for 5 min. Remove the flow clean tube.

[0071] (16) Add 2 mL of deionized water to the unused sample tube, insert the tube into the sample holder, and select run for 5 min.

[0072] (17) Detect the sample tubes by flow tracing.

[0073] Flow cytometry detection spectrum analysis

[0074] (1) First, run the Blank tubes on the instrument to detect the target cell population (lymphocytes), such as... Figure 1 As shown.

[0075] (2) Each single-color tube and the same type control were tested sequentially.

[0076] CD3 single-stain tube flow cytogram as shown below Figure 2 As shown in the left image, within the circled P1 gate, lymphocytes are clearly clustered under the FITC fluorescence channel, with CD3-positive lymphocytes on the right; in the middle image, within the circled P1 gate, lymphocytes are clearly clustered under the FITC fluorescence channel, while there are no positive cells in the APC fluorescence channel; in the right image, within the circled P1 gate, lymphocytes are clearly clustered under the FITC fluorescence channel, while there are no positive cells in the PE fluorescence channel.

[0077] CD4 single-stain tube flow cytometry diagram as follows: Figure 3 As shown in the left image, within the circled P1 gate, lymphocytes are clearly clustered under the APC fluorescence channel, with CD4-positive lymphocytes on the right; in the middle image, within the circled P1 gate, lymphocytes are clearly clustered under the APC fluorescence channel, while there are no positive cells in the FITC fluorescence channel; in the right image, within the circled P1 gate, lymphocytes are clearly clustered under the APC fluorescence channel, while there are no positive cells in the PE fluorescence channel.

[0078] CD8 single-color tube flow cytogram as shown in Figure Figure 4 As shown in the left image, within the circled P1 gate, lymphocytes are clearly clustered under the PE fluorescence channel, with CD8 positive lymphocytes on the right; in the middle image, within the circled P1 gate, lymphocytes are clearly clustered under the PE fluorescence channel, while there are no positive cells in the FITC fluorescence channel; in the right image, within the circled P1 gate, lymphocytes are clearly clustered under the PE fluorescence channel, while there are no positive cells in the FITC fluorescence channel.

[0079] CD4 is a control tube flow cytogram as shown below. Figure 5 As shown; almost no positive cells were observed under the APC fluorescence channel;

[0080] CD8 same type control tube flow cytogram as shown in the figure Figure 6 As shown; almost no positive cells were observed under the PE fluorescence channel;

[0081] (3) Sample tube analysis, the flow cytogram of sample 1 is as follows. Figure 7 As shown in the left image: the first quadrant contains CD3 / CD4 double-positive cells, which are true CD4+ T lymphocytes, representing the helper T (Th) cell subset, accounting for 22.3%; the second quadrant contains CD3+CD4- lymphocytes; the third quadrant contains CD3-CD4- lymphocytes; and the fourth quadrant contains CD3-CD4+ lymphocytes, which are not Th cells.

[0082] Right image: The first quadrant contains CD3 / CD8 double-positive cells, which are true CD8+ T lymphocytes, representing the suppressor T (Ts) cell subset, accounting for 2.38%. The second quadrant contains CD3+CD8- lymphocytes; the third quadrant contains CD3-CD8- lymphocytes; the fourth quadrant contains CD3-CD8+ lymphocytes, which are not Ts cells.

[0083] The flow cytogram of sample 2 is shown below. Figure 8 As shown in the left image: the first quadrant contains CD3 / CD4 double-positive cells, which are true CD4+ T lymphocytes, representing the helper T (Th) cell subset, accounting for 18.34%; the second quadrant contains CD3+CD4- lymphocytes; the third quadrant contains CD3-CD4- lymphocytes; and the fourth quadrant contains CD3-CD4+ lymphocytes, which are not Th cells.

[0084] Right image: The first quadrant contains CD3 / CD8 double-positive cells, which are true CD8+ T lymphocytes, representing the suppressor T (Ts) cell subset, accounting for 2.37%; the second quadrant contains CD3+CD8- lymphocytes; the third quadrant contains CD3-CD8- lymphocytes; the fourth quadrant contains CD3-CD8+ lymphocytes, which are not Ts cells.

[0085] Flow cytogram of sample 3 as shown Figure 9 As shown: Left image: Quadrant 1 contains CD3 / CD4 double-positive cells, which are true CD4+ T lymphocytes, representing the helper T (Th) cell subset, accounting for 20.49%; Right image: Quadrant 1 contains CD3 / CD8 double-positive cells, which are true CD8+ T lymphocytes, representing the suppressor T (Ts) cell subset, accounting for 2.57%. Quadrant 2 contains CD3+CD8- lymphocytes; Quadrant 3 contains CD3-CD8- lymphocytes; Quadrant 4 contains CD3-CD8+ lymphocytes, not Ts cells. Quadrant 2 contains CD3+CD4- lymphocytes; Quadrant 3 contains CD3-CD4- lymphocytes; Quadrant 4 contains CD3-CD4+ lymphocytes, not Th cells.

[0086] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A bovine peripheral blood CD4 group + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... It includes the following steps: (1) Collect whole blood from cattle to be tested using EDTA anticoagulant tubes and set up sample tubes, blank control tubes, single positive control tubes and isotype control tubes; the single positive control tubes include CD3 single positive tubes, CD4 single positive tubes and CD8 single positive tubes, the single positive control tubes are also called single staining tubes, and the isotype control tubes include CD4 isotype control tubes and CD8 isotype control tubes; (2) After adding the corresponding antibody to the sample tube and the control tube, mix well and incubate at 4°C in the dark or stain for 30 min. Mix well every 5 min during the incubation process. (3) After incubation, add red blood cell lysis buffer to lyse the cells, then centrifuge to discard the supernatant, disperse the cells, wash the cells, resuspend the cells, and prepare blank control tubes, single positive control tubes (except CD3 single positive tubes) and isotype control tubes for instrumental testing. (4) Add 30 µl of 1:100 fluorescent secondary antibody to the sample tube and CD3 single positive tube, and incubate at 4°C in the dark for 30 min. The fluorescent secondary antibody is prepared by dissolving 10 µl of Goat anti Mouse IgG1: FITC, STAR132F in 990 µl of staining buffer. (5) After incubation, the cells are centrifuged again, the supernatant is discarded, the cells are scattered, the cells are washed, and the cells are resuspended. The CD3 single positive tubes are ready for instrumental testing. (6) Add 5 µl Mouse anti Bovine CD4:Alexa Fluor 647 and 5 µl Mouse anti Bovine CD8: RPE to the sample tube, incubate at 4℃ in the dark for 30 min, and then perform the test.

2. A bovine peripheral blood CD4 group as described in claim 1 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The antibody added to the sample tube in step (2) is Mouse anti Bovine CD3, and the amount added is 2.5 µl.

3. A bovine peripheral blood CD4 group as described in claim 1 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The blank control tube, single positive control tube, and isotype control tube are mixed blood samples from all the blood samples to be tested.

4. A bovine peripheral blood CD4 group as described in claim 1 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The antibodies placed in the blank control tube, CD3 single positive tube, CD4 single positive tube, CD8 single positive tube, CD4 isotype control tube and CD8 isotype control tube in step (2) are respectively: staining buffer, Mouse anti Bovine CD3, Mouse anti Bovine CD4: Alexa Fluor, Mouse anti Bovine CD8: RPE, Mouse IgG2a Negative Control: Alexa Fluor, Mouse IgG2a Negative Control: RPE.

5. A bovine peripheral blood CD4 group as described in claim 1 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The specific procedure for centrifuging and discarding the supernatant is as follows: centrifuge at 4℃ and 500 g for 5 minutes, discard the supernatant after centrifugation, and disperse the cells.

6. A bovine peripheral blood CD4 group as described in claim 1 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The specific procedure for washing cells is as follows: add 1 ml of Flow Cytometry Staining Buffer to wash once, centrifuge at 500 g for 5 min at 4℃, discard the supernatant, and disperse the cells.

7. A bovine peripheral blood CD4 group according to claim 1 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The specific procedure for resuspending cells is as follows: add 300 µl of Flow Cytometry Staining Buffer to resuspend the cells.

8. A bovine peripheral blood CD4 group according to claim 4 + CD8 + The flow cytometry method for detecting T lymphocytes is characterized by... The amounts of antibody placed in the blank control tube, CD3 single positive tube, CD4 single positive tube, CD8 single positive tube, CD4 isotype control tube and CD8 isotype control tube in step (2) are 5µl, 2.5µl, 5µl, 5µl, 5µl and 5µl, respectively.

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