Rat splenocytes and CFSE

Hi Flowers,

I'm tring to study rat splenocytes proliferation using CFSE, but I didn't
see any proliferation in my culture.

This is the summary of the protocol:

Splenocytes were obtained after mechanical breaking up of the spleen and  red
cell lysis

25x10E6 cells/ml were incubated with 5µM CFSE in PBS/BSA 0.1% for 8
minutes/37°C

(CFSE from Fluka, 21888)

washed once with PBS/BSA 0.5%

resuspended in RPMI complete medium /10% FBS and incubated 5 minutes/37°C

centrifuged

cells were resuspended 50.000cells/ml in RPMI complete medium/ 10% FBS/
10µg/ml PHA (Irvine Scientific, 96691)

100.000 cells/200µl  in 96 well round bottomed were seeded and culture until
96h

cells were collected

washed once with PBS

acquired with FACSCanto and no differences were observed between CFSE
stained stimulated and unstimulated cells in FL-1.

is it possible I use too much CFSE to stain cells or seeded few cells/well?

Thank you for all your advices

Have a nice week

Roberta




-- 
PhD in Neuroscience
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