I am intended to perform Lymphocyte mediated serial cytotoxicity assay. I need to keep my PBMC for longer time, I am going to evaluate that for how many iterations my effectors cells (Lymphocyte) can eradicate freshly replenished targets cells. However, after first iteration I found suppression in growth of PBMC after adding the target cells for 2nd time. (Here, I wanna add that I’m sure that my effector cells can eradicate the freshly replenished targets cells for 3-4 times).

Currently, I am using medium RPMI 1460 containing 10% FBS, 2mM glutamine, 1% nonessential amino acids, 1% sodium pyruvate, and 50 U/ml penicillin and streptomycin and IL-2 100U/ml.

what kind of more cytokines, or ingredients i need to add more in my mediumt to grow the PBMC for longer time???

Thanks.