Mark_KuKuruga at BD.com
Fri Jun 25 17:10:22 EST 2004
Yes, the PI is quenching the anti-BrdU-F fluorescence. You can ameliorate
this effect by lowering the PI concentration . . . I've used 10-20 ug/ml
effectively, and you could go lower if the problem persists. You may lose
stoichiometric staining at lower than 5 ug/ml, but this still works as a
peak marker, to find G1 vs. G2/M.
Perhaps you could do a series . . . 20, 10, 5, 1 ug/ml . . . as compared to
50 ug/ml . . . to see that DNA distribution is comparable, and then look
for the BrdU-F fluorescence to increase. Remember that as PI concentration
decreases/increases, spectral spillover will change. If, however, you
"normalize" (i.e., place the G1 peak in the same channel regardless of PI
concentration), the spillover correction will be similar regardless of PI
level. Hope that's clear . . .
Colette Charland <Colette.Charlan To: Cytometry Mailing List d at uvm.edu> <cytometry at flowcyt.cyto.purdue.edu> cc: (bcc: Mark KuKuruga/SDCA/BDX) 06/25/2004 11:33 Subject: Brdu PI AM
Greetings from Vermont,
Can anyone explain why it is that Brdu Fitc positive cells
decrease from 62% to 30% after staining with PI? Is there some sort of
quenching or do the cells need to be fixed before the PI step. I
to say that our student has beautiful stimulation and wonderful PI
staining, but we cannot seem to get reproducible results when we put them
Thanks in advance for your help
Research Facility Coordinator
University of Vermont
College of Medicine-Flow Cytometry Core Lab
89 Beaumont Ave.
Burlington, VT 05405
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