Comparison of major commercially available transfection reagents using described flow cytometric method that quantifies transfection efficiency.

293T cells underwent chemical transfection using labeled or standard transfected nucleic acids (pNL4-3 or mCherry plasmids) and different commercially available transfection reagents as described in Methods. Gating on viable cells was performed as shown in Fig 1. Each experiment was performed in triplicates and six independent times (6 per plasmid and 12 in total) with specific (un)labelled nucleic acids. Data in each experiment were normalized by the average of the experimental control (standard transfected nucleic acids) in each experiment and were then pooled together. This approach increases statistical power while taking into consideration the inherent differences in transfection efficiency among different transfection methods (different chemical transfection reagents and plasmids). The non-parametric statistical Mann-Whitney test was used for comparisons between groups. Median and interquartile range (IQR) are shown. The transfection efficiency was assessed by both amount of labelled DNA (A) and protein (B). The use of TransIT-X2 and Jet Prime gave the best (and comparable) transfection efficiencies, whereas the efficiency was reduced with Lipofectamine 2000 and Fugene HD. There was a major decrease in the amount of detectable labelled DNA with Lipofectamine 2000 and Fugene HD.