In vitro Cell Viability Assay

Cell viability of HA-Ad:CD-PEI⁺:pDNA complexes in HeLa cells relative to Lipofectamine 2000 and untreated cells as positive and negative controls, respectively, was performed using the MTS assay. Cell viabilities were measured as a function of HA-Ad:CD-PEI ⁺:pDNA complex N/P ratio. For the assay, HeLa cells were cultured overnight at a density of 10,000 cells per well in complete DMEM medium supplemented with 10% FBS at 37 °C, 5% CO₂, and 95% relative humidity in 96 well microtiter plates (Greiner, 96-well multiwall plates-flat bottom). After 24 h, the cells were washed with PBS and the HA-Ad:CD-PEI⁺:pDNA complexes in serum free DMEM media were added for 24 h before addition of MTS reagent (15μL) and incubation for 2 h. The absorbance was measured at a wavelength of 492 nm using a multimode plate reader (Beckman Coulter, DTX880). The cell viability (%) relative to control cells cultured in media without any transfection agent was calculated as [A]_test/[A]_control × 100%, where [A]_test was the absorbance of the wells with PR⁺ and [A]_control was the absorbance of the control wells. All cytotoxicity values were measured in sextuplicate and averaged.