I am currently doing some CRISPR/Cas9 KO on human melanoma cell lines. I found in different articles the puromycin concentration for selection in A375 (1 μg/ml). But, I cannot find this information for the WM3211 melanoma cell line.
Has anyone ever use this WM3211 cell line for gene KO experiment?
For selection 1 microgram/ml should work with your cell line in 3-4 days. After selection, change concentration to 0.5 microgram/ml. Note that cell killing works best when cell density is low (~50%).
Hi, generally speaking puromycin selection and the concentration required is cell line dependent. If you cannot find specific literature for your cell line it is perhaps best to set up a kill curve using varying strenghts of puromycin.
Personal experience for me is that 1-5 µg/mL puromycin is suitable for most cell types.
For selection 1 microgram/ml should work with your cell line in 3-4 days. After selection, change concentration to 0.5 microgram/ml. Note that cell killing works best when cell density is low (~50%).
I would suggest doing a kill curve analysis to determine the most suitable concentration. I have seen however, 2ug/ml also works fine. If your cells are resistant to the antibiotic, they will be stable even after greater concentrations. For best results start with 500ng/ml and then gradually increase, this will lead to a better selection.
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