How to improve viability of isolated/cultured mouse splenocytes?
wmk at htmlsmith.com
Fri Jan 3 09:43:57 EST 2003
I'm assuming that 2me = 2-mercaptoethanol?
> 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>...
>>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
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