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>
>I've just started using FACS analysis for analyzing apoptosis in PBMC a
>couple of months ago. I figured that I would first try the inexpensive
>technique of PI staining. However, to date, having tried both direct
>staining with PI (with sodium citrate, Triton X-100, RNAase A) and
>ethanol-fixation prior to PI staining, I have not been able to get either
>to work reliably.
>Since my results from both techniques don't seem to correspond well with
>what I've found in literature, I've been finding it difficult to analyze.
>Any ideas or suggestions would be much appreciated.
>
My first suggestion is to look at the cells under the microscope,
preferably a fluorescent scope. PI stained cells will work well. You
should see some of the classic features of apoptotic cells: membrane
blebbing, chromatin condensation, apoptotic bodies, etc. This will confirm
that the process is occurring in your system.
Of the 2 flow methods you mention, I believe the 1st analyzes nuclei
while the 2nd is a whole cell method. I prefer the whole cell method as is
it easier to discriminate cells from debris. Generally, with PBMC we see a
clear well defined A0 peak to the left of the G0 peak. Try setting your
machine's trigger to 25% of the fluor. of the G0 peak to eliminate non
cellular events. You may also find the position of the A0 peak in relation
to the G0 peak changes with time, which may depend on how strongly you are
inducing cell death.
If you are inducing apoptosis and analyzing the cells at the correct
post fixation time, you should see a clear peak with slightly reduced fluor.
representing the apoptotic cells. I would highly recommend that you mathch
up what the cytometer tells you with what you see under the microscope.
Good luck,
Tom
--------------------------------------------------------
Thomas W. Mc Closkey, Ph. D.
Director, Flow Cytometry
North Shore University Hospital
Biomedical Research Center
350 Community Drive
Manhasset, Long Island, New York 11030
ph: 516-562-4844 [office]; 516-562-1135/4641 [lab]
6/10/97 1:16:20 PM
E-mail: thomasm@nshs.edu
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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