detaching cells but leaving matrix behind

Gys de Jongh Gys_nospam_deJongh at planet.nl
Mon May 24 14:17:42 EST 2004


"jg374 at cam.ac.uk" <jg374 at hermes.cam.ac.uk> wrote in message
news:Pine.SOL.4.58.0405211348002.8412 at orange.csi.cam.ac.uk...
> I was given a protocol to detach some CHO cells from the flask so as to
> leave the matrix behind, It was 15min at 37 with PBS(Ca/Mg free) + 5mM
> EDTA and 5mM EGTA and proteinase inhibitors. However this solution totally
> fails to detach more than 30% of the cells even if I leave it for 30 mins
> and give the flask a good bashing.  Any ideas how I can improve it's
> efficiency without affecting the matrix, which I want to solubilize in
> sample buffer?
>
Hi,
1) Wash the cells twice with PBS
2) Remove the last PBS wash

Now we found the following three treatments will romove all the cells
leaving the extracellular matrix behind :

A) UV light for about 20 min
B) Heating in an air incubator at 60 degrees for 20 min
C) Store at minus 20 degrees overnight in the deepfreeze
    one freeze/thaw cycle will kill all the cells

All three methods will give you a dish with the extracellular matrix and a few
attached , but dead , cells. The dead cells can be wased off with PBS before
seeding or will float in the medium at the first medium change after seeding.

C) works the best if cell growth on the matrix is compared
The dishes , wrapped in a plastic bag , can be stored in the deepfreeze for ever
I think. They still promoted cell growth after a couple of years

hth
Gys





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