Cytotoxicity After Transfection (Lipid Transfection)

Question

Accepted Answer

Reduced cell viability and cell death observed following transfection procedure Common causes: 1) Sub-optimal DNA to transfection reagent ratio causing toxicity 2) High endotoxin levels in plasmid DNA preparation 3) Cell density not optimal (should be >90% confluent) 4) Antibacterial agents used during transfection making cells more permeable 5) Complexes not thoroughly mixed causing concentration in wells 6) Cationic lipid reagent oxidized from excessive vortexing 7) Selection antibiotic added too soon (before 72 hr) Solutions: 1) Optimize DNA:Lipofectamine 2000 ratio from 1:0.5 to 1:5 2) Use high-quality plasmid DNA with low endotoxin from PureLink kits 3) Ensure cell density >90% at transfection 4) Do not use antibiotics (chloroquine, penicillin, streptomycin) during transfection 5) Thoroughly mix plate/wells after adding complexes 6) Do not vortex or agitate cationic lipid reagents excessively 7) Wait at least 72 hr before adding selective antibiotic for stable cell lines