apoptosis and trypsinization

Robert hewitt rhewitt at helix.nih.gov
Thu Sep 7 19:54:48 EST 1995


Dear Internetter,

I have been trying to estimate the level of apoptosis in the SW480 colon
cancer cell line by propidium iodide staining and flow cytometry.  The
result I get is that 30% of cells are apoptotic, which seems awfully 
high!

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?

Any comments would be gratefully received.

Thanks in advance,

Robert Hewitt.






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