CFSE staining

From: Roccm@mf.mri.sari.ac.uk
Date: Wed Mar 21 2001 - 10:51:53 EST


Dear Flowers and Flowerettes,

I have a question for those of you with expertise on CFSE:

I am setting up CFSE staining of sheep lymphocytes for LTT and have trouble
in obtaining a decent UNIFORM staining of my cells. In other words, the CV
of each peak (population) in FL1 is very wide (included the unstimulated
control-single peak). I had tested the same protocol in the past (8 months
ago) and at that time I had quite good results. The only difference between
then and now is that my CFSE stained cells are resuspended in a different
media AFTER the CFSE staining (IMDM instead of RPMI).
Can the change in growth media influence the amount of CFSE retained by my
cells? Or is it more likely that I'm setting my FACS wrong? or something odd
in my reagents? I load CFSE in HBSS using pluronic, CFSE final concentration
is between 2.5 an 5 micromolar, 20' of loading.
Any help will be greatly appreciated, and I'll post a summary of the answers
for the list as usual.

Thank you very much for your help,
Mara

***********************
Dr. Mara S.L. Rocchi
Moredun Research Institute
International Research Centre
Pentland Science Park
Bush Loan
Penicuik EH26 0PZ
Scotland UK
e-mail: roccm@mf.mri.sari.ac.uk
Tel: 0131-4455111 ext. 464/461
Fax: 0131-446235



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