Cells were seeded on either 24- or 96-well cell culture treated plates at a density to ensure ~80% confluent cultures at 24 hr after seeding. Typically 80,000 cells/cm2 surface area of the culture dish were seeded in cell-specific medium. Transfection using Lipofectamine 3000 was performed according to the manufacturer's protocol with a DNA to Lipofectamine ratio of 1 : 3 w/v. A transfection enhancer, the 3000 enhancer reagent (1 : 2, DNA : Reagent, w/v), was used along with the Lipofectamine 3000 transfection reagent for all transfections. Typically 100 ng and 500 ng of plasmid DNA were transferred to each well of the 96-well plate and 24-well plates, respectively. The standard complexation protocol was employed for Viromer RED according to the manufacturer's instructions. Briefly, Viromer RED was diluted (1 : 24 v/v) and the plasmid DNA was diluted independently to 18 ng/μL in the provided dilution buffer E. A 22 μL volume of diluted DNA was added to 4 μL of diluted Viromer RED and the mixture then was allowed to stay at room temperature for 15 min. For transfection, 100 ng of plasmid DNA, Viromer RED mixture, was added to each well of the 96-well plate by dispensing 6.7 μL per well. In specified experiments, 6 hr before transfection, the media was changed to either DMEM with antibiotics and without serum or DMEM with antibiotics and with serum. For quantitative evaluation of protease activity (as a measure of cell viability), luminometry, or fluorescence microscopy, cells were used 24 hr after transfection. Full paper
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