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I am optimising some panels for flow cytometry, to look and proliferation and intracellular cytokine expression in spleen and DLN cells after 3 days stimulation with antigen in culture. All is going quite well, apart from the fact that a large percentage of my cells stain for my live/dead viability stain after the 72 hour incubation. I can analyse just the live cells still, but I'm a little concerned about having so many dead cells in my cultures. After gating on lymphocytes, between 5- 15 % of my cells are alive. Is this normal? It seems worryingly high. The unstimulated controls have the highest proportion of dead cells. I have conducted a few assays lo look at adding extra media components, which did reduce the problem a little, but not completely. I have also varied cell seeding concentration and validated the stain against propidium iodide. I have stained some cells before plating also and this are very much alive (less than 1% dead) so its something that happens in the culture. Does anyone have any ideas? Also can someone tell me how well their splenocyte populations survive after 3 days in culture?
Many thanks!
