How do you select for stable transfectants in a suspension cell line? Soft agar - (May/09/2006 )

I have been struggling with this transfection for a while now = trying to stably transfect HL60 cells with a prc/cmv vector... getting good survival with 400 ug/ml G418 at the start ... but when I do the limiting dilution or soft agar, they don't grow anymore!!! What is the protocol for selection with soft agar?? Anyone?? Thank you so much

proflina

hi
the purposes of the 2 exp are not the same. First selection with G418 selects the transfected cells, whereas soft agar tells you if cells are capable to grow alone and form colonies. So i would increase slightly the G418 concentration for at least 1week supp, and then redo a soft agar test. Moreover, i'm not sure that the pressure is well done and appropriate in soft agar assay...

Oh I see.. so I don't add G418 into the soft agar? Or do I?