I am currently working on generating a knockout of the IIIG9 gene in AF22 cells using a lentiviral vector containing a puromycin resistance cassette. As part of the preparatory work, I attempted a puromycin kill curve using a starting stock of 16 µg/mL. I applied a 1:2 serial dilution down to 0.25 µg/mL, seeding the cells 24 hours prior to treatment under standard conditions (DMEM/F12 + N2/B27 + EGF/bFGF on laminin/poly-L-ornithine coating).

However, I encountered a major issue: all cells died at every concentration, including the lowest tested (0.25 µg/mL). I initially suspected Sytox Green toxicity, which I had included for cell death visualization, but control wells with Sytox Green and no puromycin remained viable. This leads me to believe that the AF22 cells may be exceptionally sensitive to puromycin.

Given this outcome, I would like to ask:

• Do you have any recommendations or internal data on the appropriate puromycin concentration range for AF22 cells?
• Would it be more appropriate to perform a new kill curve using 0.01–2 µg/mL to find the best concentration for selection?

I would be very grateful for any guidance or references you could provide.