3 replies to this topic

[laraka]

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.

[bob1]

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.

[laraka]

bob1, on 17 March 2011 - 12:58 PM, said:

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?

[bob1]

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