RE: Gating for apoptosis

Kotsianidis, Ioannis  wrote:

I am a little bit confused with the literature about the detection of
apoptosis using annexin+PI staining.


Can someone tell me which is the right gate to measure apoptosis in the
following picture (a myeloma cell line)? 


Should I select R1,R2 or both? 



Hi Ioannis,


    I think the goal of an "apoptosis assay" is really to answer the
question- what is the percentage of intact cells which are currently
detectable as undergoing the process of apoptosis by some characteristic
which I can measure?  So here are some points to consider:


    1]	So you need to examine all intact cells, while trying to eliminate
from analysis  debris and clumps.  In initially verfying your system, I
think you need some method to determine what you are gating on.
Fluorescence meicroscopy of PI stained samples is easy, and in my
experience, for cell lines the classical signes of apoptoisis are ususlly
clearly visible.  


    2]	Another option is to set up in culture a positive control adn then
sort gates R1 and R2.  


    3]	Finally i would suggest taking a look at Comparison of seven
quantitative assays to assess lymphocyte cell death during HIV infection:
measurement of induced apoptosis in anti Fas treated Jurkat cells and
spontaneous apoptosis in PBMC from children infected with HIV, AIDS Res Hum
Retro, 14: 1413-1422, 1998 which shows morphologocal changes in Jurkat cells
as reflected in LS as they undergo apopstosis.	


    So I think you need to answer the question as to gating on R1 or R2 or
both yourself.	If I was  areviewer of your paper, you would have to
convince me that the gating strategy you chose was correct, in that it made
an effort to include all cells while attempting to eliminate apoptotic
bodies and membrane fragments which could also bind annexin and give
erroneous results.  


good luck,

Tom


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Thomas W. Mc Closkey, Ph. D. 
Director of Flow Cytometry, North Shore University Hospital 
Assistant Professor of Pediatrics, New York University School of Medicine 
Boas Marks Biomedical Research Center, 350 Community Drive 
Manhasset, Long Island, New York 11030 
ph:  516-562-4844 [office], 516-562-1135/4641 [lab]  fax:  516-562-2866 
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