PFA fixation and dead cells

McCloskey, Thomas thomasm at nshs.EDU
Fri Nov 11 14:49:31 EST 2005


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From: paolo [mailto:paolo+ at pitt.edu] 


I work with HIV infected cultures and I need to determine apoptotic vs live
vs dead cells. I use an Coulter XL with a single laser (488nm) and I have to
fix cells with 1% PFA as they are loaded with HIV. 

Hi Paolo,
    I see you've already received lots of responses, but let me add a few
comments from a different angle.  If you are looking at primary lymphocytes
from HIV infected persons or at cell cultures which you 've infected in
vitro, I think it is wel established now that HIV causes cells to die via
apoptosis.  In other words, cells are not dying by necrosis,  or other
pathways of cell death.  
    With that said, all cells in your cultures are either live or apoptotic.
So, biologically,  there isn't really a third category of "dead",   Once
cells have progressed down the apoptotic pathway past the point of no
return, they are committed to die.  
    There are lots of assays which allow fixation and which are applicable
to quantify apoptosis in your cultures, I refer you to:  Mc Closkey TW,
Chavan S, Tamma SML, and Pahwa S, 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.
Good luck,
Tom
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* 
Thomas W. Mc Closkey, Ph. D. 
Scientist in Charge, Flow Cytometry 
The Institute for Medical Research, North Shore University Hospital 
Assistant Professor of Pediatrics, New York University School of Medicine 
Biomedical Research Center, 350 Community Drive 
Manhasset, Long Island, New York 11030 
ph:  516-562-4844 [office], 516-562-1084 [lab] 
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