Activation of 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 or PMA + 1 μM
ionomycin (final concentration) in the presence of a
protein calcium inhibitor such as Golgi plug containing brefeldin
A ( ng/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
(eBioScience), vortex and incubate for 10 min at RT
in the dark.
5. Spin 5 min, 500g.
6. Aspirate the supernatant,
7. Continue with staining after adding
50μl PBS to each tube.