Hey all, I'm trying to select fibroblasts after transduction with shRNA to integrin a5 but I don't know why, my cells are not dying after treatment with puromycin. I treated even cells not transfected and they didn't die. There would be something in culture media that could interfere and give them resistance to this antibiotic? I already prepared new puromycin stocks and treated them but they are still 100% live.
Before starting a selection you need to establish at which concentration of Puromycin (or any selection agent) your untransfected cells die. That can also vary with the Puro batches. Basically, plate cells at non-confluent/low density for a few hours until they are attached and then add your medium with Puro at different concentrations (e.g. 0.5 ug/ml, 1 ug/ml up to 5 or 10 ug/ml). Change the medium every other day (or as you would do with your cells you want to select) and see at which concentration they die. That's the one you want to use for selection. Also, if the cells are confluent, they usually won't die, so you can split them into your selection medium and the non-transfected will start dying.
Hi Wanessa, this is not my field of expertize, but to eliminate the possibility of serum factors interfering with puromycin, you could set up an experiment with serum and serum-free cultures and add puromycin.
So it would be 1) serum-free cells without puromycin and 2) serum-free cells with puromycin. 3) serum containing media without puromycin and 4) serum containing media with puromycin.
Than you all for your answers! I'll try to follow both recomendations and after I'll give you some feedback.
Thank you.
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