apoptosis and trypsinization

Alex Morla amorla at midway.uchicago.edu
Thu Sep 7 11:28:49 EST 1995


In article <Pine.SGI.3.91.950906200529.19352A-100000 at helix.nih.gov>,
rhewitt at HELIX.NIH.GOV (Robert Hewitt) wrote:

> I wonder whether I may be inducing apotosis when preparing the single cell
> suspension.  The procedure I use is to trypsinize, resuspend in DMEM + 10%
> FBS, spin at 700 rpm for 5 mins in a Sorvall benchtop, resuspend in PBS
> without calcium or magnesium, count, then spin again, then resuspend in an
> appropriate volume of PBS, before fixation.  All this is completed in
> between 40 and 60 minutes.  The whole procedure is done at room
> temperature to avoid "cold shock" induction of apoptosis. 
> 
> Is it likely that I could be inducing apoptosis by the above procedure?  
> If so, then would there be evidence of apotosis given the fact that cells 
> are fixed within 60 minutes of trypsinization?  

I don't know about induction of apoptosis by this method, but I do know
that some cells are very sensitive to being washed in PBS without
proteins. Try adding 0.5% BSA to the PBS (if it doesn't interfere with
your assay) you use in washes, this may help maintain the viability of
your cells.

Alex Morla
-- 
Dr. Alex Morla
Dept. of Pathology
The Univ. of Chicago                                              
                            
                                                                          



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