How would you investigate inconsistent transfection efficiency across cell lines?

Why

Use the same well-characterized reporter plasmid, then measure the fraction of viable reporter-positive cells rather than only the total fluorescence of the well. Record cell identity, density, confluence, passage, mycoplasma status, plasmid quality, DNA-to-reagent ratio and the time between delivery and readout. A highly fluorescent culture might contain a small fraction of bright cells rather than high transfection across the whole population.

Titrate reagent and DNA while monitoring toxicity. A membrane-damaging delivery protocol can increase reporter signal among survivors while selectively killing sensitive cells. Normalize carefully for viable cells and compare an appropriate mock-transfected control.

If one cell type remains refractory, explore an alternative validated delivery modality and measure transgene expression and function separately. Report the chosen condition and performance distribution per cell type rather than declaring one reagent universally optimal.