I'm trying to do a stable transfection of hybridoma cell line NG108-15. I'm using FuGENE(R) 6 transfection reagent in ratios of 3:2 (reagent:plasmid) in 6 well plate and in confluence of 70%. After two weeks in G418 (250 ug/ml), all my cells are dead. Does anyone have any suggestions?
Decrease the reagent and then G418 dosages. in first passage after transfection wash the cells completely with PBS+10% DMSO
Did you linearize your plasmid before transfection?If not i recommend doing that and also trying different combinations of Fugene reagent and your plasmid?Like Babak suggested try reducing your G418 too because the dose you are using might be toxic to the cells.
the common dosage of G418 for selection of transformants are 400 mic/ml and higher. So I think the problem is in transfectin. Howmuch plasmids did you use for transfection?
I have three suggestions, the first is to titrate the G418 doses, the second will be to perform two sequential transfections in order to increase the efficiency of getting stably transfected cells and finally start the selection after only 72h. In ourt lab we are using Dreamfect Gold reagent for producing stably transfected cells.
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