Question (2326)
Question
Why did all my cells die during puromycin selection after viral transduction in my CRISPR experiment?
ABM community
Verified customer
Asked on Jan 29 2026
Answer
Complete cell death during puromycin selection most commonly indicates one or more of the following: puromycin concentration is too high for the specific cell line or selection was initiated too early, before sufficient expression of the puromycin resistance gene. To optimize selection, abm recommends the following:
1. Perform a puromycin kill curve- Test a range of puromycin concentrations (e.g., 0.25–10 µg/mL, depending on the cell type).
- Identify the lowest concentration that kills all untransduced control cells within 3–5 days.
- Use a slightly lower concentration (typically 0.5×–0.75× of the kill-curve dose) for post-transduction selection.
- Allow 48–72 hours after transduction before adding puromycin.
- This recovery period allows expression the puromycin resistance gene.
- Include a positive control lentivirus (e.g., GFP-only or selectable marker–only) to verify infection efficiency.
- Use an appropriate MOI for the target cell type.
- Include a transduction enhancer (e.g., polybrene at 4–8 µg/mL, if compatible).
ABM Scientific Support
Answered on Jan 29 2026