I have infected NK-92 cells with lentivirus at MOI 30 using protamine sulfate and spinoculation (2,000 rpm for 1 hour, ~600 × g). The following day, I replaced the medium, and after 48 hours, I confirmed GFP expression. At 72 hours post-transduction, approximately 10–15% of NK-92 cells expressed GFP.

I then began puromycin selection at 0.5 μg/mL and 1 μg/mL for 48 hours, followed by another 48-hour selection. However, all cells in the transduction group died, while a few cells in the negative control group remained alive.

For reference, a WST-8 assay indicated that 2–3 μg/mL puromycin kills around 50% of NK-92 cells within 48 hours. Using those concentrations in previous trials resulted in complete cell death after a few selection cycles, so I have been trying lower doses.

When using the same lentivirus to infect HEK293T cells, I successfully obtained stable GFP-expressing cells after puromycin selection.

I am confused about what I might be missing and would appreciate guidance on optimizing transduction efficiency and survival during selection.