Very few cells are fluorescent — transfection efficiency is extremely low (Transfection)

Question

Accepted Answer

Microscopy shows scattered positive cells; flow cytometry confirms positivity rate is very low (< 5 %). Common causes: 1) Cell density was not optimal (too high or too low at the time of transfection) 2) Reagent-to-DNA ratio was suboptimal 3) Cell type is intrinsically refractory to chemical transfection 4) DNA amount was too low 5) Expression window observation was too early Solutions: 1) Target 70 – 80 % confluence at the time of transfection 2) Run a reagent : DNA ratio gradient (e.g. 1:1, 1:2, 1:4 µL reagent per µg DNA) 3) Switch reagent or method (electroporation, viral delivery) for hard-to-transfect lines 4) Increase plasmid amount within recommended range 5) Extend expression window to 24 – 48 h before scoring