Ix83 inverted motorized microscope

Mitotracker CMTM Orange (M7510, Thermo Fisher Scientific) was used as per the manufacturers protocol. Briefly, a stock solution of Mitotracker was reconstituted to 1 mM with DMSO. The stock solution was then diluted to a working concentration of 100–500 nM in pre-warmed complete medium. Medium was removed from cells and 1 ml of Mitotracker staining solution added. Cells were incubated at 37°C with 5% CO₂ for 30 min. Cells were then washed once in complete media or were fixed and stained as previously described.
For analysis of mitochondrial morphology 10 random fields of view (FOV) were taken of three separate wells for each cell type after Mitotracker staining using an Olympus IX83 inverted motorized microscope. Images were analyzed blind and scored by eye for any cells that demonstrated any tubular branching mitochondria and were reported as a proportion of all cells per FOV.

The microscope slides containing the stained samples were imaged using an UPLFLN 40x oil immersion objective (Olympus America, Center Valley, PA) and digital CCD camera ORCA-R2 C10600-10B (Hamamatsu Photonics, Shizuoka Prefecture, Japan) mounted on an IX-83 inverted motorized microscope (Olympus America, Center Valley, PA). Fluorescence images of ten randomly selected fields of view per slide were taken at 40x magnification (6.22 pixels/μm resolution) for each sample.