I have a frozen splenocytes and want to proliferate them in cell culture to study intracellular cytokines? Do you have experience and what shoud I add to RPMI 1640? How long I need to stimulate them?
I am assuming that you would like to measure T cell proliferation and measure cytokines in the supernatant. Like Ronald suggested, you need to use complete RPMI-1640 (10% FBS + antibiotic +/- beta mercaptoethanol) supplemented with recombinant IL-2 + your antigen.
The culture time frame would depend on your assays. If measuring cytokines, 24-48 hour incubation is recommended. But you need to leave the cells for longer to see T cell proliferation (4-7 days after CFSE staining).
If you have the ability to get/work with fresh splenocytes, that would be the best. I have seen really good responses with fresh splenocytes which went to absolutely no response once the splenocytes were frozen.