Preparation of Mammalian Cells (HEK293T) for Transfection and Fluorescence Confocal Microscopy

HEK293T cells (100000 cells) were cultured in a 35 mm imaging dish with a glass bottom and an imprinted 50 μm cell location grid (Ibidi; cat. no. 81148) in DMEM medium supplemented with 1% (v/v) sodium pyruvate, 1% (v/v) antibiotics, 1% (v/v) glutamine, and 10% (v/v) fetal calf serum (FCS). JetPEI (PolyPlus; cat. no. 101-10N) was used to cotransfect cells with pmTurquoise2-Mito and Cox8-RamR-FLAG constructs. Sixteen hours post-transfection, the cells were washed with phenol-red free, serum-free, antibiotic-free RPMI imaging medium (ThermoFisher; cat. no. 11835030) containing 1% glutamine. Bodipy625 was diluted in the imaging medium to a final concentration of 450 nM. After 15 min of incubation at 37 °C, the free dye was washed away with the imaging medium. Cells were imaged live at 37 °C by confocal imaging, and their positions on the grid were marked. Subsequently, the cells were washed with PBS, fixed with 4% paraformaldehyde (PAF) for 15 min, and permeabilized with 0.1% Triton-X100 in PBS for 5 min. Cells were immunolabeled with mouse IgG1 Anti-Flag antibody (Sigma; cat. no. F1804) overnight at dilution 1:200 in PBS. Next, cells were washed with PBS and labeled with secondary donkey anti-mouse antibody conjugated to Alexa Fluor 568 (ThermoFisher; cat. no. A10037) at dilution 1:400 in PBS for 30 min. Finally cells located at the stored positions on the grid were imaged by confocal microscopy.