Table 2.

Troubleshooting common issues in viral transduction

Problem	Likely cause	Quick fixes
Low % positive cells	Insufficient activation; weak virus; no contact enhancement via polycation	Verify activation. Use fresh, concentrated supernatant; add polycation; multiple spinoculation;
Good % positive but low expression	Promoter not T-cell-friendly; position effects	Try EF1α/PGK/MSC-family promoters; include an easily detectable reporter/surface tag for QC
High toxicity after transduction	Excess polycation; prolonged exposure; residual transfection reagent	Reduce polybrene; shorten exposure; promptly replace media
No improvement with higher MOI	Supernatant quality or route is limiting	Concentrate by PEG/ultracentrifuge; move to Retronectin-coated plates; split doses rather than one large hit. PEG precipitation of 24-48 h harvests is a simple approach.