I had more them 20 replies to the question below, so I thought I would
summarize briefly:
Many people reported similar problems with no real solution. Others
suggested longer incubations or not growing the E.C. to confluence.
This last suggestion would work, but was not an option for me. Others
suggested Dispase or other enzymes. I have not tried all the enzymes yet.
What DID work was to first rinse the monolayer with D-PBS without
Calcium or Magnesium, then apply a small volume of Trypsin EDTA (~1ml
for a T25 flask) and remove it immediately, then add more trypsin EDTA
and decant that immediately too.
The cells in the remaining thin layer of Trypsin EDTA round up and come
off with 2-3 minutes. These are mostly single cells, and since the
Tryspsin/EDTA is such small volume, I do not wash the cells before replating.
The brand of Trypsin/EDTA I used was MEDIATECH Cat#25-052-CI (0.05%
Trypsin, 0.53mM EDTA). I tried one other brand which did not work at
all. (!)
Mike
Michael Herron wrote:
>
> All,
>
> I am working with a transformed endothelial cell line. I have to
> culture them to confluency before checking their fluorescence by FACS.
> When I use trypsin/EDTA the cells come off nicely, but they remain in
> large "sheets" that do not come apart with pipetting as many cells will.
>
> Does anyone have any suggestions as to how to disaggragate these sheets
> of cells?
>
> Thanks,
> Mike
>
--
_____________________________________________________
/ Michael J. Herron, U of MN, Dept. of Entomology /
/ herro001@umn.edu /
/ 612-624-3212 (lab) St. Paul, MN 55108 /
/____________________________________________________/
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