2.12. Transient transfection

HEK293T cells were transfected at 70% confluence using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Plasmid DNAs were incubated with Lipofectamine 2000 in OPTI-MEM (Invitrogen) for 20 min at room temperature (20-25 °C). Plasmid-Lipofectamine mixtures were then incubated with cells in OPTI-MEM at 37 °C in a CO₂ incubator for 5-7 h. Afterwards, DMEM containing 20% FBS was added to reach a final FBS concentration of 10%. Because cotransfection of Tom22 or ANT with BK_Ca significantly decreased the expression of BK_Ca, we first transfected BK_Ca constructs to allow their expression followed by a second transfection of Tom22 or ANT 24 h later. All constructs were transfected with 10 μg plasmid/100 mm dish with the exception of Tom22 that was transfected at 1 μg plasmid/100 mm dish in experiments involving truncated BK_Ca channels. After 48 h from the second transfection, cells were collected and lysed for further analysis.