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G418 killing curve


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#1 laraka

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Posted 17 March 2011 - 11:29 AM

Hi,

I am trying to make a G418 killing curve for Hela S3 cells (untransfected). I seed the cells 8000/well in 96-well dish and after 24 hrs, add the antibiotic from 50 ug/ml to 2 mg/ml. It doesn't kill the cells. Recommended concentration for selection is generally 800 ug/ml for Helas. I was wondering if anyone working with G418 observed anything like. I'll be glad to hear your suggestions.

Edited by laraka, 17 March 2011 - 11:35 AM.


#2 bob1

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Posted 17 March 2011 - 12:58 PM

How long do you leave the cells in antibiotic for? It should take 8-10 days or so. Some cells may be left in the wells; if you look closely you will see that they are very likely senescent.

#3 laraka

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Posted 17 March 2011 - 09:53 PM

How long do you leave the cells in antibiotic for? It should take 8-10 days or so. Some cells may be left in the wells; if you look closely you will see that they are very likely senescent.


I was incubating for 24 hrs and then measuring the cell viability since other antibiotics that I've been using (puromycin and blasticidin) cause a massive cell death after 1 day. For g418, I extended the incubation period up to 3 days. Even though I see some granulation in cells, they still keep dividing and since they are untransfected, that surprises me.

Are you suggesting 8-10 day treatment for transfected or untransfected cells?

#4 bob1

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Posted 20 March 2011 - 04:17 PM

Both... it takes time to work so that you have enough cell death to measure.




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