Staining with PI & BrdU

Maribel Leong Maribel_Leong.RMH_FacMed at muwaye.unimelb.edu.au
Thu Mar 28 04:43:32 EST 1996


Hi all!

Just a quick query from a new flow-er (we've just purchased a FACS Calibur 
after months of deliberating!! PS:  thanks for all of the replies that I 
received a while back with regards to B-D vs Coulter)...

I've been staining cells with BrdU & getting about 60-80% incorporation 
after a 2h pulse - is this the norm?  The cycling time of these cells is 12 
h.  I've tried adding PI to look at DNA content & find that 48% of the cells 
are in S phase.  Do these results make sense?  I'm not a DNA person so I'm 
not quite sure.  Someone that I spoke to thought that it would make sense, 
but my supervisor doesn't think so.  So, I thought I'd ask the opinion of 
people who do this everyday!

Also, if anyone could tell me how to make up the PI for DNA content studies, 
I'd be much appreciative.  It seems that people just use their stock (5x) 
which is diluted in Triton X-100 forever!

Any help would be appreciated.

Thanks in advance,

Maribel Leong
Dept Medicine (RMH)
University of Melbourne
Australia 



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