Quantifying Mitochondrial Morphology Changes

HeLa cells transfected with ^mtGFP or MEF^mtGFP cells were seeded at a density of 100,000 cells/well on glass‐bottom dishes (MatTek) coated with poly‐D‐lysine (Sigma) or rat tail collagen 1 (GIBCO). Cells were then treated as indicated and images were recorded with an Olympus IX71 microscope with 60× oil objective (Olympus), a Hamamatsu C8484 camera (Hamamatsu Photonics), and HCI image software (Hamamatsu Photonics). Quantification was performed by blinding the images and then scoring cells based on the presence of primarily fragmented, tubular, or elongated mitochondria, as before (Lebeau et al, 2018 (link)). At least three different researchers scored each set of images and these scores were averaged for each individual experiment and all quantifications shown were performed for at least three independent experiments quantifying a total of > 60 cells/condition across all experiments. The data were then prepared in PRISM (GraphPad, San Diego, CA) and plotted on a stacked bar plot to show the average morphology and standard error of the mean across all experiments. Statistical comparisons were performed using a two‐way ANOVA in PRISM, comparing the relative amounts of fragmented, tubular, or elongated mitochondria across different conditions.