T cell activation for intracellular cytokine detection by Flow Cytometry - (Aug/13/2008 )
I will start a series of experiment for intracellular detection by flow cytometry of IFN-g and IL-2 in sorted primary T cells that have been stimulated for 8 days with different compounds.
I was planning to activate (at Day 8) my T cells with PMA and Ionomycin for additional 6 hours in the presence of Golgi-Stop before permeabilization and staining.
I am actually screening the literature trying to figure out if there are established concentrations of PMA-Ionomycin that would fit with my experimental set up, but I would be very grateful if I could get any suggestions from you too. I would be interested to know which concentration to use for primary T cells, to be sure not to kill my cells, or not to misinterpret my results.
For a pilot experiment I have used quite a high concentration of both the stimulants, just to see if I was able to get any results, and I have used 100ng/ml PMA and 4uM of Ionomycin, and I got quite a nice staining of both the cytokines of interest.
Thank you very much!
Why use PMA/iono at all, are your T cells defective in TCR proximal signaling? Use anti-CD3 stimulation instead, this better simulates what actually happens in vivo. You would also only have to optimize one condition as opposed to two.
In any T cell stimulation assay you should do some kind of titration to see what concentrations and times are best suited to your study.
You also didnt specifiy what subset of T cells you have in culture. The gamma-interferon and IL-2 cytokine profiles are not the same for different T cell subsets (ie. CD8s vs Th1s, Th2s etc.)