I have transiently transfected 67NRs, seeded in a 96 well plate at a seeding density of 15000 per well, using Lipofectamine 3000. 24 hours after transfection, I observed that there are atleast 50% GFP positive cells, indicating the transfection efficiency. I tried to puromycin select the untransfected cells. I did a kill curve (0-10ug/ml) concentration. However, even at the higehst concentration am not observing significant cell death even in the untransfected positive control well. Though I did observe the cells rounding up after 24 hours of puromycin treatment, there is no visible cell death even after 72 hours. Please provide your valuable sugegstions, so that I could improve my puromycin selection protocol for 67NRs.