Hi
I am growing HepG2 cells expressing NTCP receptor at a high level under 500ug/ml of G418
At the same time I want an efficient knockdown of one gene and ready to do Puromycin selection.
I am wondering if the combination of antibiotics will bring synergistic cell death and risk of loosing majority of cells after double selection.
Should I drop G418 and only do puro at first place and add G418 in selected cells.
If anyone has done a similar experiment, please share your expertise.
Thanks and Regards, Ramesh
I wouldn't think it a problem as long as you've determined kill curves for each antibiotic in the parental cell line. We have selected various epithelial cells with G418 (1mg/mL) and hygromycin (4ug/mL) without issues.
Thanks Thomas for sharing your experience and dose titration is a good suggestion.
Your G418 concentration (1mg/ml) is quite optimal. Wondering hygromycin at 4ug/ml (which is multifold lesser than the standard dose) can induce any DNA double strand break in eukaryotic cells.
Just to save time I will start G418 selection of Puro survived cells.
Cheers!
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