Mouse Treg detection procedure

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

2. Label surface antigens according to cell surface antigen staining methods. 0.125 μg FITC anti-mouse CD4, 0.06 μg APC anti-mouse CD25 antibody was used in the 100 μl system of CD4 and CD25 antibodies according to the instructions. It is best to follow the detailed instructions for these reagents. Incubate at 4 °C for 30 minutes. An Fc receptor blocker is added prior to incubation of the surface antibody.

3. Wash the cells with a pre-cooled flow staining buffer solution (centrifugation and transfer).

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

5. Incubate in the dark at 4 ° C for 30 minutes to 18 hours (incubation for 30 minutes to 18 hours, the same result).

6. Centrifuge the cells by centrifugation with 2 ml of Permeabilization Buffer (1:9 deionized water) and discard the supernatant.

7. Repeat step 6 to wash the cells.

8. (Optional) Add a disruption buffer containing 0.5 μg of Fc receptor blocker to ensure that the 100 μl system is incubated for 15 minutes at 4 °C.

9. The blocking solution was not washed away. The system was supplemented with 0.5 μg of PE antimouse/rat Foxp3 antibody and PE Rat lgG2a isotype control (diluted with Permeabilization Buffer working solution) and 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.

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

11. Repeat the previous step to wash the cells.

The cells were resuspended in an appropriate volume of Flow Cytometry Staining Buffer and detected and analyzed on-board. 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.

The above steps are for reference only and are subject to the manufacturer's instructions.