sathy at vasci.umass.edu
Tue Oct 27 02:47:55 EST 1998
We are also currently trying to stain mouse splenocytes with
CFSE dye (cells are stained with 2.5-5 uM final conc. dissolved
in DMSO final <0.1% in PBS for 8-10 min @ room temp. and
washed 3-5 times with 10% serum medium ) to analyse cell divisions
of CD4 cells and intracellular cytokine production, but with less success.
We have a number of problems in the assay. The main problem was
that splenocytes stained with CFSE did not respond to stimulation with
either ConA or PMA/Ionomycin or in some instances tend to die in
culture. Did anyone else have this problem in CFSE staining assays?
We would appreciate if anyone can give any suggestions to get around
this problem. We also had the problem of compensating FL2-FL1
to distinguish different cell generations (peaks). Does anyone have
any suggestions for compensation of FL2-FL1 or do we have to try other
flourochromes such as Red670 and TriColor (FL3 on FACScan) for
Thanks in advance.
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