Hello Shamsa Saleh

Did you perform the "kill curve" for puromycin?

You should perform a kill curve experiment for puromycin to determine the minimal concentration that effectively kills the non-transduced PBMCs. Antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic (puromycin) to determine the minimum concentration of an antibiotic that can kill all the cells in a specific period of time.

This is a crucial step before using a selection antibiotic to kill non-transduced cells. It is recommended to perform separate kill curve experiments for different cell and antibiotics because each cell type differs in antibiotic sensitivity.

So, first determine the minimal puromycin concentration required to kill all the non-transduced PBMCs. Use a range starting from 0.5–10 µg/mL of puromycin on the cells.

You may follow the protocol provided in the link below.

https://manuals.cellecta.com/lentiviral-construct-packaging-and-transduction/v10a/en/topic/calculating-a-kill-curve

Best.