Although Annexin-V plus PI is good for detecting apoptosis, as you
know, this method can not tell the difference between necrosis and late
apoptosis. We also experienced high background staining on some cells or
cell lines using Annexin-V conjugates. Recently, we think we have developed
a sensitive method that specifically looks at apoptosis which can be used
with either adherent and suspension cells (for flow cytometric analysis or
microscopy). If you are interested in testing this method in your system,
please contact Chris Brotski at email: chrisb@biosource.com. We could send
you samples.
Jimin
Jimin Wang, Ph.D.
Sr. Scientist
Biosource International
WWW.biosource.com
-----Original Message-----
From: Tom Mc Closkey [mailto:thomasm@nshs.edu]
Sent: Friday, February 11, 2000 1:55 PM
To: cyto-inbox
Subject: Re: Annexin V (AV) and propidium iodide (PI) staining to
identify apoptotic/necrotic cells]
> Martin Kelly wrote:
> >
> > > we would like to ask some advice r.e. the interpretation of some
> > work we have been doing on induced sputum and BAL from normal
> > subjects and COPD patients. We have been using 'flow' to look at
> > the granulocytes, and examine the AV and PI staining in the
> > events which we have gated on, which we feel are in the region
> > in which one would expect to find granulocytes. Cells coming up
> > AV+PI- we interpret as apoptotic; AV+PI+ as necrotic.
>
Bear in mind that late stage apoptotic cells will no longer
exclude
PI. The best identification for the process of aopoptosis is
fluorescence microscopy after labeling with a DNA stain [PI, DAPI, AO,
etc.]. You should be able to identify those cells undergoing apoptosis
and determine if "necrosis" is actually present in your system.
Alternately,run a 2 nd assay such as TUNEL or subdiploid assay to
verify
your annexin results.
Regards,
Tom
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*
Thomas W. Mc Closkey, Ph. D., Director of Flow Cytometry
North Shore University Hospital, 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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*
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