I isolated CD4+ CD24- T cells by Dynaebeads, after evaluatig their purity, I cultured them in RPMI supplemented by AntiCD3/28,10%FBS ,but after3days I found 2 cell populations in flowcytometry.As I know there were not dead cells how I can confirm all of them are sames? is there any size changes during invitro culture?
T cells before activation are relatively small cells with very little cytoplasm. But once activated they become big and the FSC vs SSC plots differ slightly from university activated cells.
@siva Gandhapudi,Tnx for your response, but I see 2 populations that are separated from each other? is it possible that during stimulation some of them gain different size and granularity?
Can you post the FSC vs SSC plots, which will give a better idea. Yes it's possible to see two distinct plots as the blast cell get bigger and will be in monocyte gate. You dont get 100 % T cell activation in this type ( anticd3/cd28) of cultures unless u hit them with high antibody concentrations. So you will see activated and non activated cells. Also how are u checking for dead cells? It's unlikely that you don't have any after 3 days as the activated T cells will start dying in this culture unless they are supplemented with il2.
Depending on the concentration of anti-CD3/CD28 beads, but the 2nd population might be blasts as previously mentioned, but blasts should be larger in FSC but the same SSC.
Another case could be that the cells may still be attached to the beads when undergoing flow cytometry, thus it may move the cells up in terms of SSC but should not increase their FSC much.
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