Calcein-AM/CoCl2 assays

mPTP opening was assessed by calcein-AM/CoCl₂ assays as described previously.⁴⁷ Briefly, the isolated CMs were incubated with 5 μM calcein-AM for 30 min at 37°C and then incubated with 40 μM CoCl₂ for 20 min at 37°C. When the mPTP was unopened, calcein fluorescence was maintained in mitochondria in the presence of CoCl₂. When the mPTP opened, calcein was lost from the mitochondria. Fluorescence intensities were determined by confocal microscopy.