Mitochondrial Dynamics in MEF and HeLa Cells

wt and Drp1-null mouse embryonic fibroblast (MEF) cells^{48 (link)} and human cervical adenocarcinoma (HeLa S3) cells were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO₂ atmosphere. Before imaging, the cells were plated at 50–60% confluency in glass-bottom microscopy dishes (35 mm, N° 1.5 glass, 14 mm well-diameter, MatTek, Ashland, MA) pre-coated with fibronectin/gelatin solution (10 μg/mL/20 μg/mL). Cells were transfected with 1–2 μg of plasmid DNA encoding either mEGFP alone or the mEGFP Drp1 fusion constructs together with the mCherry-Mito-7 plasmid (gift from Michael Davidson, Addgene plasmid #55102) using the Lipofectamine LTX kit (Invitrogen) according to the manufacturer’s protocol. Cells were incubated for 24–48 h post-transfection. One hour before imaging, the culture medium was exchanged with Fluorobrite DMEM media (Gibco) supplemented with 1X GlutaMAX and 10 mM HEPES, pH 7.2 (Gibco).