Quantification of Mitochondrial Superoxide Production

To assess the production of superoxide by mitochondria, MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific) was used. At 2 days post‐transplantation, cells were incubated with MitoSOX Red (5 μmol/L) at 37°C for 10 minutes. Cells were then washed 3 times with sterile PBS. Using a fluorescence plate reader, MitoSOX Red was excited at 485 nm and fluorescence emission was measured at 590 nm. With 10 observations in each group, fluorescence values were compared among the 2 groups of independent transplantation experiments and a control group (Figure 5).

Mitochondrial superoxide production. H9c2 cells were exposed to MitoSOX Red reagent 48 hours after mitochondrial transplantation to measure superoxide production. MitoSOX Red was excited at 485 nm, and fluorescence emission was measured at 590 nm. Whisker box plot shows distribution of fluorescence readings for 10 measurements per group. No statistically significant difference was observed in the population's mean of the post‐transplant groups vs control. Fluorescence measurements of MitoSOX‐treated groups were normalized to the untreated control and are displayed as bar graphs, for the mean±SD. Symbols: White line denotes the population median, “x” indicates the population mean, and filled black dots represent outliers.