Mouse splenocytes dying after 3 days in culture - (Feb/03/2012 )

Hi everyone,

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!

What kind of media do you use? I have done T cell differentiation in vitro and they have stayed alive for at least 5 days. However they were purified CD4 T cells. I have done spleen cell stimulations in vitro and but have harvested them after 2 days. In all cases I used RPMI (FCS+PenStrep+Glutamine+Hepes+Na pyruvate+ 2ME). Not sure whether this will help.