Currently I'm working on IGF-1 activity on T-cell marker experssion. I isolated mouse splenocytes and treated cells with 100 ng/ml IGF-1, splenocytes were cultured in RPMI 1640 plus 10% FBS, however 48 hours later almost all cells died. Anybody knows how to dear with such condition? any suggestion?
It's usual situation for splenocytes - some of them have tendency to die after isolation. Did you included untreated controls - maybe IGF-1 helps cells to survive?
I same time, in our experience we've found cell death is not equal for CD3+ and CD3- cells. So adequate gating strategy during flow cytometry analysis could help to unmask effect of IGF-1.
Without stimulation (PMA/Ionomycin, CD3/CD28, IL-2), splenocytes will quickly die.
It's a regulatory mechanism to prevent aberrant T cell activity in the body.
Culture the splenocytes overnight with PMA/ionomycin, rinse the cells, then do your treatment.
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