HepG2 cells were transfected with a luciferase expression plasmid using the designated reagents at the manufacturer’s recommended reagent-to-DNA ratio indicated beneath each bar. Transfections were performed in 96-well plates using 0.1 µg of plasmid DNA per well. Luciferase expression (bar graph) and lactate dehydrogenase (LDH) levels (line graph) were measured at 24 hours post-transfection. LDH levels are reported as % cytotoxicity compared to cells alone and were measured using a commercially available colorimetric assay; all values at or below zero are represented as zero on graph.

HeLa cells were plated in 12-well plates and transfected in parallel at 50% and 90% confluency. TransIT®-LT1 Reagent (LT1) transfection were performed in duplicate using 1 µg of a luciferase expression vector and 3 µl of reagent per well. Lipofectamine™ 2000 (LF2K) transfections were performed using 1.6 µg of a luciferase expression vector and 4 µl of reagent per well. Twenty-four hours post-transfection, cells were harvested and assayed for luciferase activity. The data represents the average luciferase activity from three experiments performed on different days.

To quantitate the effect  of the TransIT®-LT1 (LT1) and Lipofectamine™ 2000 (LF2K) transfection reagents on cell viability, we used the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). This assay measures the levels of ATP in a cell population. It is the most sensitive,cell viability assay available. HeLa cells at 50% and 90% confluency were transfected in duplicate using 3 µl of TransIT®-LT1 Reagent (LT1) or 4 µl Lipofectamine™ 2000 (LF2K) per well. Twenty-four hours post-transfections the cells were harvested, and cell viability determined using the CellTiter-Glo® Luminescent Cell Viability Assay. Data was compared to untransfected HeLa cell control and represented as a percentage of the control. The data from three experiments performed on different days was averaged. For a detailed discussion of cytotoxicity markers and assays, please see the following paper: Use of Multiple Assay Endpoints to Investigate the Effects of Incubation Time, Dose of Toxin, and Plating Density in Cell-Based Cytotoxicity Assays.