In article <CK4qt2.II5 at sci.kun.nl>, theos at sci.kun.nl (Theo Schoenmakers)
wrote:
>> Dear netters,
>> There was a discussion on letting PC-12 cells attach here recently. I
> have quite a similar problem, and I hope someone can give some hints on
> how to resolve it.
>> I am working with NG108-15 cells, a hybridomal cell line of a rat
> neuroblastoma and a mouse glioma. These cells were fine, growing
> quickly and attaching with no problem on culture dishes till about a
> month ago. After we took them out of the liquid nitrogen after the Xmas
> holidays, nothing is working out.
>> The cells have a very hard time attaching to culture dishes. Coating
> the dishes with collagen doesn't really seem to help much. Lately I had
> one lot that was attached reasonably. However, after changing the
> medium for one with 10 uM prostaglandin E1, 1 mM theophylline and 1%
> Foetal Calf Serum (instead of the usual 10%) in order to start a
> differentiation, the cells let go of the support after two days. This
> treatment did not pose any problems a month ago.
>> WHAT IS GOING ON?
>> Does anyone have an idea? Suggestions? Please....
>> Thanks a lot,
> Theo Schoenmakers
I had a similar problem with BC3H-1 cells. Although they were fine in
culture flasks for subculturing, the monolayers would eventually begin
lifting from multi-well plates we used in assays of endogenous nicotinic
receptors. I got a great tip from John Merlie that I slightly modified
that overcame this problem. We now routinely plate our BC3H-1 cells FOR
ASSAYS into multiwell plates that have been coated with denatured porcine
skin gelatin. However, we carry the cells in culture flasks as usual
WITHOUT attachment to gelatin.
The protocol is:
1. Make up a 2% solution of porcine skin gelatin (Sigma sells the stuff)
in
water.
2. Autoclave.
3. Dilute to 0.2% for use on plates. Add more than enough to cover the
bottom
of the plates and incubate 3 hours at 4 degress C.
4. Remove and use (no drying necessary) or store at 4 degrees C. We've
stored
for up to five days with no apparent problem.
We eventually do see detachment of BC3H-1 cells from our assay plates even
with this protocol (I don't know why. Some selective pressure probably).
But it takes about 6 months and we just thaw some more. I should say that
our assays are fairly rigorous with respect to cell attachment.
Good luck,
Duke
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