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Cell Death after selection pressure has been removed?

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Cell Death after selection pressure has been removed?

Postby Ilsalmi » Mon May 19, 2008 2:24 pm

I have an adrenergic receptor stable transfected CHO cells - I have been culturing them in both with and without AB (for quarantine purposes) - and the cell population in media without all antibiotics (incl. the selection ones - G418 and Hygromycin) is dying.
There is no bacterial nor Mycoplasmal contamination, no fungus either - all triple checked by appropriate tests. The culture media is clear and there are still few attached cells (approx 30%) and I keep splitting them, but a large number of cells are floating and dying.
The culture with antibiotics was fine.
It doesn't seem to make sence - why would they get worse when the selection stress is removed?

Thanks for all input.
Ilsalmi
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Re: Cell Death after selection pressure has been removed?

Postby daniel.kurz » Tue May 20, 2008 9:15 pm

The only things that I can suggest is you have some kind of receptor protein on the cell membrane or the agar you are growing the cells on lacks a molecule that is required by the cell. I would try another agar base and see if you get any better results. The way I read your message you are getting mitotic developments so it must be an issue of dietary or necrosis. I would try using a new agar and buy new agar to make sure it hasn't gone bad and no cross contamination has occurred. I would also recommend that you get your hands on some new petri dishes or glass ones because plasticizers like bisphenol-A can leach out and cause odd responses to cells by bonding to the cell receptors. You can try those in any order you like depending on which is most cost and time effective. Good luck and give us an update on what happens. I am curious.
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Re: Cell Death after selection pressure has been removed?

Postby Ilsalmi » Wed May 21, 2008 8:54 pm

Thank you, unfotrunately (fortunately) it is no mystery after all.

I have screened the several batches of heat inactivated serum i have been using, and one of them turned out to be just way bad. The same one I used for making up media without ATB.
The paralel culture with selection antibiotics was in good batch serum.
Next time I won't trust even big name suppliers:(

Thank you again.
Ilsalmi
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