Recently, I did a kill curve study for my NPCs. It was found that 0.5ug/ml was fine for the cells to die within 3-7 days? Now, I wish to ask can I challenged my NPCs (which are transduce with the lentivirus with Puro resistance gene) starting from 0.5 ug to 10ug/ml (say) gradually? Since my cells are transduce do they can now be resistance to what ever dose of puromycin will be or their is a certain limit above which I can't go?
Again, my kill curve showed a dose of 0.5ug/ml was enough to kill the cells by day 3-7.
Many thanks.
Hello. There is definitely an upper limit above which you cannot go. If 0.5ug/mL worked previously, the next step would be to treat each your NPCs without the puromycin resistance gene and the NPCs transduced with the puromycin gene with 0.5ug/mL puromycin at the same time, and another concentration (maybe 0.7ug/mL) if you want to possibly save some time in case 0.5ug/mL isn't sufficient this time. Once your NPCs without the puromycin resistance gene die, you should have NPCs with the resistance gene still surviving. At this point, you can assume with a degree of confidence that your cells are stably transfected (or transduced). However, you should check for expression of your gene of interest by PCR and western blot to be sure. You should also continue to culture them for a few days to a couple weeks in that same concentration of puromycin. This will allow them to adapt to the puromycin and your gene of interest and achieve a degree of signaling stability. Your stably transduced cell line(s) should then be useful in producing some great experimental results.
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