Hela cells and stable transfection help please! - (Oct/12/2006 )
I am trying to transfect Hela cells, but I can't seem to get stable cell lines. I have tried CaPO4 and am currently trying lipofectamine 2000. I can get transients with both methods, but when I select with G418 (14 days) the number of positive cells(by ICC) is getting less and less with each trypsinization. I don't suspect the insert is toxic since it is just a modification of another vector that we have in a stable cell line.
I am new to transfections so any help would be appreciated
Thanks in advance!
I am new to transfections so any help would be appreciated
Thanks in advance!
may b the conc. of G418 is on the higher side so the cells expressing low levels of resistance gene r dying.
I doubt that is it. We are only using 0.5 mg/ml G418. Our other cell line is stable even in 1.0 mg/ml G418.
i know that the stably transfected cells can gradually kick out the plasmid over a period of time. May b thats what is happening with this particular cell lines
Maybe try virus... I always use virus for stable cell lines
u always use virus to make stables ? is that much easier ?
That's the only way I know....
pBabe vector.... package in phoenix cells, and infect mouse cells... then I got stable cells
If you want the protocol, I can send you one.