No subject

Sathiyaseelan 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 
other staining.

Thanks in advance.

Sathi
UMass-Amherst





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