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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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My cells won't die!
Posted by: Winn-Dixie (IP Hidden, New member, 1)
Date: June 5, 2005 03:55PM
I'm trying to set up stable transfections of Panc-1 cells. I linearized my vectors, then transfected using Gencarier-1. The transfection efficiency is around 80% by lacZ staining with very low cell death. After 3 days the cells were split 1:50 into 24-well plates in medium containing 1 mg/ml Geneticin (dissolved in PBS, frozen in 1ml aliquots). The cells seemed to start dying after a few days, but now, over a week later they just won't die and most wells are completely overgrown. The Geneticin concentration I got from a paper using the same cells and vector. Any idea anyone?
Re: My cells won't die!
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 6, 2005 09:47AM
it sounds as if something is wrong with the geneticin itself. has it been stored for a long period of time? 1mg/ml should have been sufficient to kill the cells.
Re: My cells won't die!
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: June 7, 2005 07:03PM
Have you done a killing curve on parental PANC-1 cells first to determine your optimal slection conc.?
1mg/ml Geneticin seems to me is too high for most cells?
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