Flow cytometry analysis

1. Stimulate murine T cells with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml ionomycin (Sigma) in RPMI medium for 4 hr.

2. For the last 3 hr, add 5 µg/ml Brefeldin A (Sigma).

3. Collect cells and wash with PBS/0.3 % BSA at 300 rpm, 4 °C for 8 min.
   

4. Stain cell surfaces with the respective antibodies in 100 µl PBS/0,3 % BSA for 20 min at 4°C in the presence of 100 µg/ml 2.4G2 (anti-FcγRII/III; purified from hybridoma supernatants) to reduce unspecific antibody binding. Chemokine receptors were stained for 45 min at 4 °C.

5. Wash with PBS at 300 rpm, 4 °C for 8 min.
   

6. Perform fixable dead cell staining with 500 µl PacificOrange-NHS (1µg/ml, Life Technologies) for 20 min at 4 °C.

7. Wash with PBS at 300 rpm, 4 °C for 8 min.
   

8. Fix cells with 500 µl of 2% paraformaldehyde (dissolved in PBS) for 20 min at room temperature.

9. Wash with PBS/ 0.3 % BSA at 300 rpm, 4 °C for 8 min.
   

10. Stain intracellular proteins in 200 µl of 0.5% saponin (in PBS) for 30 min at room temperature.

11. Wash with PBS/ 0.3 % BSA at 300 rpm, 4 °C for 8 min.

12. Dissolve cells in PBS/ 0.3 % BSA.

13. Acquire on a suitable flow cytometer.

For intracellular staining of transcription factors perform steps 1-7.

  8b. Fix cells with 500 µl FoxP3 fixation buffer (eBioscience) for at least 1 hr at room temperature.

  9b. Wash with PBS/ 0.3 % BSA at 300 rpm, 4 °C for 8 min.

10b. Perform intracellular staining in 100 µl of 1x FoxP3 permeabilization buffer (eBioscience) for 30 min at room temperature.

11b. Wash with PBS/ 0.3 % BSA at 300 rpm, 4 °C for 8 min.

12b. Dissolve cells in PBS/ 0.3 % BSA.

13b. Acquire on a suitable flow cytometer.

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