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the events with low SSC and low FSC may depend on
>whether you have FSC and SSC amps set to log. If this is so then what you
are
>really seeing is debris form your sheath fluid.
These events are unlikely to be debris from the sheath fluid. You
could test this poossibility by running some sheath as a sample. However,
devris is very common in a populaiton of cells which are dying. So it more
likely is simply debris from the tube [apoptotic bodies, fre e nuclei,
mitochondria, membrane fragmants etc.]
>
A DNA stain is more useful. If you are doing apoptosis work then you
>may want to discriminate the necrotic cells from the apoptotic cells. Most
>people use PI to do just that.
>
Just to clarify this point, PI used in low concentaration for a
short incubation time on unfixed cells will differenttiate early apoptotic
[intact membrane] from late apoptotic or necrotic [lost membrance
integrity]. This won't help with the cell/debris resolution problem. My
recommendation was to use PI on fixed cells to stain DNA and thus
differentiate cells from other events based onn DNA content.
Also if the events are a contaminating cell population [ie rbc in
pbmc] , the correct combination of McAbs will allow you to gate on the cells
you're interested in.
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]
4/25/97 9:01:19 AM
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;
Web