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?
Hi Tugba,
I used to culture rat splenocytes.
We added 10% heat inactivated Foetal Bovine Serum to the medium.
Depending on the type of cell we were interested in (T-cells or NK-cells) we cultured them with IL-2 (25U/ml for T-cells and 1000U/ml for NK-cells).
We also added 3,5 µl/ml beta-mercapto-ethanol.
With kind regards,
Ronald
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.
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