NIH-3T3 (ATCC catalog # CRL-1658) mouse embryonic fibroblast cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) with 1% Penicillin Streptomycin (10 3 U/mL) solution in a humidified incubator containing 5% CO2 at 37 °C. Before the imaging experiment, cells were seeded in a 35mm glass bottom dish with 10mm micro-well, #1 cover glass fibronectin-coated confocal dishes. For experiments, cells were used between passages #6-18. The imaging of the live NIH-3T3 cells was carried out by a laser scanning confocal fluorescence microscope, using 60X oil immersion objective Olympus FV-3000-IX-81. The Mito-pH2 dye fluorescence images were recorded with band path 580-620 nm upon excitation at 561 nm.