stable line making of adherent cells - concentration of puromycin for adherent cells (Jul/18/2006 )
once again i am stuck ...i am trying to make a stable line out of adherent keratinocytes, using puromycin selection. i am using 1microgm/ml concentration, and after 72 hrs, i still see living cells..
i am working with this cell line for first time. your suggestions as to how much concentation of puromycin shud be ideally used for adherent cells will be greatly appreciated.
from my experience, the best thing to do is a kill curve because different cells respond differently to the antibiotic. select about 5 concentrations of puromycin, add them to the cells and watch for cell death over a period of a week. this way you can choose the best concentration that kills your cells after a few days. clontech describes it very nicely in their protocol for using the tet-on system. i use about 2 ug/ml pur for my particular cells (SMC). hope this helps.
2 to 10 µg/ml should be applied.
Go to cayla website, you'll find on section antibiotics/puromycin some guidelines for certain cell types.
The first thing you should do is testing what your cell can endure. .Especiallly for the differernt cells.