Measurement of mitochondrial superoxide production

After the treatments, isolated mouse cardiomyocytes were loaded with MitoSOX Red (5 μM, Thermo Fisher Scientific, USA) in cardiomyocyte plating medium or perfusion buffer (+ CaCl₂ 1 mM, BSA 0.5%) and incubated at 37 °C for 20 min. MitoSOX Red specifically targets mitochondria in live cells. The more superoxide production in mitochondria, the greater is the fluorescence intensity observed. After 20-min incubation, cells were washed two times with cardiomyocyte plating medium. The live cell imaging on cardiomyocytes was done using an Axio Observer Z1 motorized microscope (Zeiss, Oberchoken, Germany) with a 10× objective. Red fluorescence intensity in an individual cardiomyocyte was quantified using ImageJ (NIH), which indicated mitochondrial superoxide production.