Foxp3 Intracellular staining in whole blood

1. To 100 μl whole blood add anti mouse CD3^FITC, CD4^APC, CD25^PE or mouse isotype^PE (1 mg/ml). Mix on Vortex and incubate for 20 min at RT.

2. Add 2ml of Pharmlyse (Becton Dickinson), vortex and incubate for 10 min at RT in the dark.

3. Spin 5 min, 500g.

4. Aspirate the supernatant.

5. Add 100 ml of Fix solution A (Caltag Fix and Perm Kit), mix on vortex and incubate for 15 min at RT.

6. Add PBS and spin 5 min, 500g.

7. Aspirate the supernatant.

8. Add 100 ml of Perm solution B (Caltag Fix and Perm Kit). Mix on Vortex and incubate for 15 min at RT.

9. Add PBS and spin 5 min, 500g.

10. Aspirate the supernatant.

11. Add anti mouse Foxp3^PB (0.5 mg) or mouse isotype^PB (0.5 mg) to cell pellets. Mix on Vortex and incubate for 30 min on ice.

12. Add PBS and spin 5 min, 500g.

13. Aspirate the supernatant.

14. Add PBS <0.5ml.

15. Collect >25,000 events.