[Cytometry] effect of mitogen

snehal mhatre snehalrm at gmail.com
Mon May 2 11:52:40 EDT 2011


We want to study effect of different concentrations of mitomycin C (MMC) on
lymphocytes. so for this purpose, we separate PBMCs, and culture them for
72hrs with different concentrations of MMC. and then check viability of
cells with PI. for baseline, we check viability of cells on day 0. but after
72hrs, might be due to culturing of cells, the FSC vs SSC scatter is not
proper. so it is difficult to gate lymphs and hence checking viability is
not specific, as we are not sure whether we are gating specific population.

so my queries are

1) for culturing we just adjust cell count with complete media (RPMI-1640)
and add MMC. Do we have to take any specific precautions during this
procedure, to maintain the cell morphology
2) Will adding a marker like CD45 will be of help?
3) Should we use 7-AAD instead of PI?

Thanks
--
Regards
Snehal Mhatre
PhD student
NIIH, Parel
Mumbai


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