apoptosis and trypsinization
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
Dr. Alex Morla
Dept. of Pathology
The Univ. of Chicago
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