double stable transfetcion - (Aug/27/2007 )

Hey everyone,
I ran into a problem recently when trying to do a stable transfection of HEK293 cells. I'm trying to do a double stable transfection. I managed to do the first stable transfection successfully by selecting with G418 (.5 mg/mL). I'm running inot problems with the second transfection. I'm selecting the second transfection using hygromycin using the concentration I got from doing a kill curve (.2mg/mL). 24 hours after transfection I add medium containing the G418 and the hygromycin. When I look at the cells the next day, a majority of the cells (>90%) appear dead (round floating). Even with the kill curve at .2mg/mL of the hygromycin it took about a week for me to see major cell death. Is it possible that my cells have become more sensitive to hygromycin after transfection? Any insight into what is going on would be great.

Thank,
Joe

Can you select for only hyg first, then add neo later on?

24h after transfection is little early to add antibiotic. I would go for 48h test.

I agree that 24h is too early to start the selection process, so you may first split your cells in approriate ratio for selection (1:10, 1:20 or 1:50). Then you can add appropriate concentration of hygromycin that you have determined to the media 48h posttransfection.

we would start selection for clones between 48hrs-72 hrs and we also dilute the cells like 1:25 and 1:50

Hey everyone,
So I'm still having trouble with my hygromycin selection. Since selecting with 200ug/ml has been killing my cells (cells are round and detached from flask) off in a mere 2-3 days I tried lowering the concentration to 50ug/mL. Even with such a low concentration, the transfected cells are dieing with in 2-3 days. Anyone have any insight into why this is happening? I've also tried adding hygromycin at later time points (24, 48, 72 hrs) and the cells still die.

Any help would be appreciated
joe