How to improve viability of isolated/cultured mouse splenocytes?

[Willem Kuhtreiber]

Hi,

I isolate/culture mouse splenocytes in the following way:
Mash spleen through a cell strainer (40 micron mesh) in PBS, lyse red 
blood cells with NH4Cl and put in culture in DMEM with 10% FBS.

Trypan Blue immediately after the isolation gives viabilities better 
then 85% - 90%, but after 24 hr culture the viability has gone down to 
30% - 50%.

I need the splenocytes "untouched' in flow cytometry studies, i.e., I 
cannot stimulate with things like ConA.

I tried a whole bunch of things with cell density, culture additives, 
incubation temp, etc, without much avail.

Does anybody know how to improve this?

Thanks for your input!

Willem Kuhtreiber, Ph.D.
wmk at htmlsmith.com