Good day!I am currently generating stable cell lines( KON ) using TTA vector and G418 as drug selection agent.?After two weeks i got isolated colony from some wells at the 900ug/ml (G418). I tried to expand the cells, but it seems like they are not growing well "Slow growing to non growing". Any recommendation to trouble shoot my problem...
Thank you in advance.
Hi,
What size flask are you using to expand the cells?
Also, it might be too much G418. I usually use 400 or 500 ug/ml.
Thanks so much
Angel
Concentration may be a factor but take into account that when you isolate colonies you should nod dilute. Keep cells as much concentrated as possible and do not touch (even do not move the flask) them in 2-4 days.
Tge cells are in 96 well plate well... The cells are too few thats why i did not transfer them in to 48 well plate..
96 well plates should be ok, but with little liquid and without moving for 2-4 days.
But then another question. When you look at them: they are dead or they die when you try to take them from plates. Taking cells from 96 well plates is not easy (if "your" cells attach to the plastic; if they grow in suspension not a problem).
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