Massive cell death after transfection — reagent toxicity is too high (Transfection)

Question

Accepted Answer

Cells become rounded and detached after transfection; CCK-8 shows a sharp drop in viability vs. mock-transfected control. Common causes: 1) Transfection reagent dose is too high or inherently toxic 2) Cell line is particularly sensitive to this reagent 3) Complex incubation time on cells was too long 4) Old medium not replaced after the transfection step Solutions: 1) Lower reagent dose, or switch to a lower-toxicity reagent (e.g. Lipofectamine 3000 → 2000, or PEI alternatives) 2) Shorten the complex incubation time on cells (10 – 20 min for fast protocols) 3) Replace with fresh medium 6 – 8 h post-transfection 4) For PEI, try different molecular weights (25 kDa vs 40 kDa) or different reagents entirely