Human PBMC regulates T cell detection steps

1. Prepare PBMC and adjust the cell concentration to 107/ml.

2. Add 100 μl of prepared cell suspension to each flow-through sample tube. The number of cells is approximately 1x106.

3. Mark CD4 and CD25 according to the cell surface antigen staining method. Determine the amount of antibody (generally 20 ul, depending on the batch) according to the instructions and the label on the antibody tube. Blank tube, control tube, compensation tube and sample tube are set according to individual requirements. Incubate at 4 °C for 30 minutes.

4. Wash the cells with pre-cooled PBS solution, centrifuge at 300-400 g for 5 minutes, and remove the supernatant.

5. Dilute the fixed/broken membrane concentrate with 3 times the volume of the fixed/broken membrane dilution solution to prepare a fixed/broken membrane working solution. Note that it should be freshly prepared, and the amount of each sample is 1 ml.

6. After vortexing and resuspending the cells, add 1 ml of Fixation/Permeabilization (diluted 1:3) and vortex again.

7. Incubate at 4 ° C for 30 minutes to 60 minutes.

8. Dilute the membrane buffer with deionized water 1:9 to make a working solution. The amount of each sample is 4~5ml.

9. Without washing, directly add 2 ml of Permeabilization Buffer to wash the cells by centrifugation at 300-400 g and discard the supernatant.

10. (Optional) Repeat step 9 to wash the cells.

11. Resuspend the cells by adding 100 ul PBS.

12. Add 2% rat serum to 2 ul (closed optional) and incubate at room temperature for 30 minutes in the dark.

13. Add appropriate amount of Foxp3 antibody to the system, and add appropriate amount of isotype control to the control tube. The specific dosage should be incubated at 4 °C for at least 30 minutes. It is recommended to conduct a preliminary experiment to find the optimal concentration of the antibody.

14. Centrifuge the cells by adding 2 ml of the ruptured membrane solution and discard the supernatant.

15. Repeat the previous step to wash the cells.

16. Resuspend the cells with a suitable volume of Flow Cytometry Staining Buffer and test and analyze them on the machine. Note: Due to the use of cell fixation and membrane rupture during the staining process, there will be some changes in the morphology of the living cells. Therefore, the FCC/SSC diagram needs to be adjusted when the ring is closed.

Note: The above instructions are for reference only. Please refer to the manufacturer's instructions for specific experiments.