How to culture stable cell lines? - (Apr/13/2006 )

Hi,
I am very new in culturing stable cell line. Recently I ask a kind of stable cell line from someone. I thraw them and let them grow about 10 days and suddenly realize that I shoud add G418 to it. So I add 300ug/ml G418 to the cell immediately. Could anyone tell me is that OK or it will cause me lose the stable expressed gene? How long it will take for G418 to kill the cells that do not express the gene stably?How to maintain the gene stably expressed in cell? Should I add G418 immediatly when I thraw the cells or wait for several days to let the cell recovery and then add drug?


Thank you very much!

si your cell line on some sort of selection pressure?

If you were asked to add G418 in your culure medium, most probably your cells were transfected with your gene of interest alongwith a neomycin resistance cassette. So addition of G418 will enable only cells with gene of your interest to grow and others will die. I think 10 days is too long a period to wait before selecting for neomycin resistance. You might be lucky to find any cells with gene of your interest. It might be a good idea to start over and add G418 within one or two doubling times of the cells at the latest. Usually in the absence of a selecting pressure, the cells tend to lose constructs fast.