Hello everyone,
I have problems with HEK293 cell line and its IC50 value. In literature, 200-500 ng/ml is suggested as g418 concentration. To be convinced, I performed kill curve(in 96-well plate, 600-60 and 800-80ng/ml, 72h) 2 times and IC50 value was nearly 100 ng/ml in both, it was very low value. There was mycoplasma contamination in HEK293 and this cell line was treated with plasmocin and now I add prophylactic to protect cell line against mycoplasma. I thought that mycoplasma--tratment--prophylactic can cause such a low value. We started stable transfection(in 6 well plate) but after 9 days unransfected cells do not die at 100ng/ml. What could be the reason for such a situation.
Thank you......
Purely from a G418-perspective I'd say your cells are fine and behave as expected.
If you treat your cells with ~100ng/mL I'm not really surprised your untransfected cells are still not dead.
We have several stably transfected HEK293 cell lines in our lab and those are usually maintained at a G418 concentration of 100-300 µg/mL.
With every stable transfection we attempt, we first test our current batch of HEK293's to see what G418 concentration kills all cells in 1 week. Usually that's around 200-500 µg/mL. We select at higher concentrations and once the line is stable we maintain at lower concentrentrations. For example, select at 400 µg/mL and maintain at 200 µg/mL.
Is this of any help?
Thank you for your answer,
this time I started with 500 ug/ml and maintained at 250 ug/ml and HEK293 cells died successfully.
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