Why can't I select for puromycin-positive MCF7 cells?

Hi Gal, I am wondering that are your MCF7 cells died or not died at all after puromycin treatment?

I have some suggestions here: if all of the MCF7 cells are died, may be 2ug/ml is too high. You can try less concentration from 0.5ug/ml. I used 1ug/ml for my MDA231 cells. Or maybe the transfection didn't work in your MCF7 cells. It's better to optimize the transfection.

If all of the MCF7 cells are alive after puro treatment, one possibility is that: when the immortalized MCF7 cell lines was made, it had puro-resistance already. So please check the information about this cell line that you are using. If not, optimize the concentration of puromycin in normal MCF7 cells. Usually the working concentrations of puromycin for mammalian cell lines range from 1 to 10 μg/ml.

Hope these answers help you. Good luck!

Best,

Shan

The concentration of puro might be different for different cell lines. We usually do a dose curve in wild-type cells first. Choose the concentration that could kill about 95% cells for positive-cell selection.