As soon as you remove the splenocytes from their in vivo environment they
start dieing, this is a normal process. To keep them alive you need to
stimulate them with specific growth factors. These factors depend on the
particular cell population you want to maintain in culture -- IL-2, ConA,
Ca/Ionophore, 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>
