intracellular cytokine staining - re-stimulation of activated T cells (Jul/10/2011 )

Hi everyone
I am doing ICS on activated T cells. How long should I stimulate the splenocytes with the whole protein antigen (since a specific T cell epitope is not defined) for re-activation of T cells? At what point should I add the brefeldin A?


Thanks a lot!

Follow the protocol. That's it.

Stimulate your cells by plate-bound anti-CD3 + soluble anti-CD28 or by PMA/Ionomycine and incubate for 4-5hours. Brefeldin have to be added for last 2 hours of incubation.