Releasing adherent cells w/o trypsin

wetsel_r at wetsel_r at
Wed Feb 17 07:44:40 EST 1993

In a previous article, adavies at (Anthony Davies) wrote:
>In article <1993Feb8.135444.2010 at>, bma14432 at writes:
>> Dear Netter,
>> I vaguely recall a conversation a couple years ago where someone
>> mentioned a protocol for releasing adherent cells from tissue culture
>> flasks/dishes without trypsinizing them. I believe it involved EDTA but
>> I'm not sure. If anyone has a reference or protocol for this I would
>> be very appreciative. My main concern is to release the cells with
>> minimal disturbance to the membrane. If you have other ideas please feel
>> free to suggest them. Thanks in advance.
>Scrape them off ???
>Anthony Davies
>Institute of Virology
>Oxford UK
Yes, I do it all the time as I need to periodically look at the expression 
of surface markers after various treatments on adherent cells.  Thus, I 
can't use trypsin (or shouldn't) when doing FACS analysis.  What I use is 
PBS with 5mM EDTA pH'ed to 7.2 after the EDTA is in solution.  1) aspirate 
media, 2) rinse with this solution, aspirate again, and 3) add fresh 
solution (2ml for T25, 4 ml for T75).  It obviously doesn't work as fast as 
Trypsin/EDTA but you should see cells lifting off within 3-5 minutes and 
you may have to smack the flask to help dislodge them.  Another suggestion 
from my grad-school mentor was to trypsinize the cells but wash them and 
resuspend them in complete media (same one used for growth), and put them 
in a poly-propalyene (sp?) tube (so they have no opportunity to attach), 
gas them with 5% CO2, and leave them be at 37'C for an hour - ideally to 
let the membrane turnover - although I've not yet tried this for FACS.

I hope some of this helps...

+  David L. Haviland, Ph.D.	     Internet:"haviland at"   +
+  Washington Univ. School of Med.   A.K.A : The Compiler                 +
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