Measuring Mitochondrial Calcium Levels

We used Rhod-2/AM (R1244, Thermo Fisher Scientific, USA), which demonstrates increased fluorescence upon Ca²⁺ binding in the mitochondria, to determine mitochondrial free Ca²⁺ [27 (link)]. Briefly, after washing samples twice with fresh PBS, dye (5 µM Rhod-2/AM) was slowly added along the sides of the single muscle fibers, followed by incubation in the dark at 37 °C for 30 min. After incubation, the glass slide-mounted Rhod-2/AM-loaded fibers were washed with fresh PBS three times (20 s/time, 1-min process). The slide was then quickly placed on the microscope stage, and the fibers were focused in the bright field (20-s process) and scanned via laser confocal microscopy in combination with an Olympus FV10-ASW system (Japan) under 594-nm krypton/argon laser illumination, with fluorescence detected at 618 nm. Analysis and statistical methods were similar to those used for the measurement of cytoplasmic Ca²⁺ mentioned above.