How to improve viability of isolated/cultured mouse splenocytes?

[Vaccine-Man]

Mouse cells need a little higher osmolarity than DMEM provides. Try
using Click's medium (or RPMI-1640 bumped to 315 mOsm with NaCl from
its 290 mOsm) and only 5% FBS. Also include 2me at 50 uM. Presumably
you're using the standard goodies in the medium - L-glutamine, sodium
bicarb, etc.

Willem Kuhtreiber <wmk at htmlsmith.com> wrote in message news:<aui9kk$hsr$1 at pcls4.std.com>...
> 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