transfecting and selecting EL4 cells - (Jun/10/2010 )
I have recently infected some EL4 cells with endotoxin free DNA using effectene. This should have introduced resistance to Hygromycin B into the infected cells.
however, after being put on selection (400 ug/ml Hygro B which is what we usually use for all other cell types) the control cells have not shown any sign of stress or dying. 10 days later the controls are now looking stressed but so do my infected cells. Has anyone infected EL4 cells with a reasonable efficiency?
i have since set up a kill curve from 200 ug/ml to 1 mg/ml of hygro B thinking that maybe the concentration of the antibiotic wasn't high enough. 48 hrs later we were expecting the cells with 1mg/ml to be at least stressed but they look fine. Any idea's on what concentration of Hygro B would be best?
Really appreciate any help
Are you sure the antibiotic hasn't gone off? 1 mg/ml is a lot for HygB.