2.8. Transient transfection

For HEK293 transfection 70% confluent cell plates (Corning 100 mm, Culture Dish #430,167) were transfected with Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MS, USA) according to the manufacturer's instructions. Briefly, for each plate 25 µl of Lipofectamine 3000 were diluted in 1.5 ml of Opti-MEM Medium (Gibco, Thermo Fisher Scientific, Waltham, MS, USA). Separately, master mix was prepared by diluting DNA (12,5 µl [1 µg/µl] /sample) plus P3000™ Reagent (25ul/sample) in 1.5 ml of Opti-MEM Medium. Master mix was then added to diluted Lipofectamine 3000 Reagent (1:1 ratio) and incubated for 15 min at room temperature. DNA-lipid complex was dropped onto plated cells. Cells were incubated at 37 °C and collected 48 h after transfection for subsequent analysis.

For fibroblast transfection 60% confluent cell plates (glass bottom dish 35 mm, Ibidi, Germany) were transfected with Mirus Bio™ TransIT™-LT1 Transfection Reagent (Thermo Fisher Scientific, Waltham, MS, USA) according to the manufacturer's instructions. Briefly, for each plate 1 µl of plasmid (1 µg/ul stock) were diluted in 1.0 ml of Opti-MEM Medium (Gibco, Thermo Fisher Scientific, Waltham, MS, USA). Then 3 µl of Mirus Bio™ TransIT™-LT1 were added to the mix and incubated for 30 min at room temperature. DNA-lipid complex was dropped onto plated cells. Cells were incubated at 37 °C and analyzed 48 h after transfection.