Interleukin Intracellular staining in activated whole blood

1. Dilute whole blood 1:1 volume to volume (e.g. 100 μl:100 μl) with RPMI1640 medium and mix well.

2. Add cell activator or mitogen to the diluted blood e.g. 50 ng/ml PMA + 1μg/ml calcium ionophore in the presence of brefeldin A (20 mg/ml).

3. Vortex briefly to mix. Aliquot 200μl into 12 x 75 plastic tubes. Incubate for 4-6 h in 5% CO2 at 37˚ C.

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

5. Add PBS and spin 5 min, 500g x2.

6. Aspirate the supernatant x2.

7. Add 50μl PBS to each tube.

8. Add anti mouse CD3^FITC and CD4^APC or mouse isotype^APC (1 mg/ml) to cell pellets. Mix on Vortex and incubate for 20 min at RT.

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

10. Aspirate the supernatant.

11. Add 100 ml of Fix solution A (Caltag Fix and Perm Kit) and incubate for 15 min at RT.

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

13. Aspirate the supernatant.

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

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

16. Aspirate the supernatant.

17. Add anti mouse IL17^PE (0.5 mg) or mouse isotype^PE (0.5 mg) to cell pellets. Mix on Vortex and incubate for 30 min on ice.

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

19. Aspirate the supernatant.

20. Add PBS <0.5ml.

21. Collect >25,000 events.