How to improve viability of isolated/cultured mouse splenocytes?

Willem Kuhtreiber wmk at htmlsmith.com
Fri Jan 3 09:42:35 EST 2003


Thanks,

I'll give that a try!

Willem Kuhtreiber

Vaccine-Man wrote:
> 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




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