David_C_McFarland@sbphrd.com wrote:
> A concept that a lot of people seem to miss is that AnnexinV is more
> than useless when used alone, it is misleading. You have to use it in
> conjunction with a dead cell discriminator. Dead cells stain very brightly for
> Annexin V. And no, AnnexinV+/PI+ double positives do not imply late apoptosis
> or apoptotic death. The PI is only used so that you don't count those cells as
> apoptotic. They are simply dead and that is all you can say about them.
We use annexin without a dead cell discriminator [ie assessment of membrane
integrity] in combo with phenetypic and/or cell cycle measure. Our goal is to
quantify the total % os apoptotic cells in a certain poplation at a given time.
Annexin alone answers this question. To exclude those cells which are late
apoptotic [annex+PI+] or "simply dead" would change the question which you are
answering. In a known cell system with a known pathway of induction of cell death,
the presumption can be made [and proven via earlier time course experiments] that
those c ells died via apoptotic mechansims. Please see AIDS Res Hum Retro 14:
1413-1422, 1998 for assay comparison data and Clin Diag Lab Immunol 8: 74-78, 2001
for application of the annexin assay.
> Also,
> timing is everything. Controls
> are paramount. And finally, try to correlate your results
> with another assay. I find that
> too often experimenters doing flow neglect the microscope.
These are all valid points. The biological question being posed must consider
the kinetics of this process and the fact that these assays yield a snapshot in
time. Negative and positve controls aare essential and I often recommend multiple
methods. and finally it is definitely time to dust off that microscope :-) and
actually look at the cells!
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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