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