Starting a stable cell line?

I've done this several times with transfected cells and I think the reason it's usually vague is that there's not a whole lot more to it other than adding the appropriate concentration of antibiotic and waiting. I'll give you a couple tips I've learned from my own experience though:

1) Never let the cells reach confluence while you are selecting for transfected cells. You want to keep them all rapidly dividing to give the growth advantage to cells expressing your gene. If you're using a serial dilution, then you'll likely find a good dilution after transfection where the cells are not too confluent too soon after beginning treatment.

2) After exposing the cells to antibiotic for 48 h (the time needed for normal cells to double twice in my case), I would thoroughly trypsinize the cells, inactivate the trypsin, and let the cells re-attach in the same flask for 4-5h, then wash off the weakly attached or non-attached cells. The idea is that non-expressing cells can double twice in the presence of antibiotic but after that, they can not make new protein (in the case of G418), so they will not be able to re-attach.

3) Be sure to change your media regularly, even if the cells have not used up all the nutrients, the antibiotic will likely be inactivated after a couple days.

Hope that helps.

James David Londino

This helped me. Although the technique you need isn't exactly the same it might help. Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

http://www.jove.com/video/50171/generation-stable-human-cell-lines-with-tetracycline-inducible-tet-on

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