Quantifying Apoptosis in Cardiomyocytes

Early apoptosis and late apoptosis/necrosis were detected using an Annexin-V FITC/PI Apoptosis Detection kit according to the manufacturer’s instruction. Briefly, cardiomyocytes were seeded into a 12-plate at a density of 5×105/well, after the indicated treatment, adherent cells were enzymatically digested for 50S with 0.25% trypsin and collected together with floating dead cells. Approximately 1×106 cells were washed twice with cold PBS, and then resuspended in 200μl cold 1×binding buffer containing 5μl of Annexin V-FITC and 5μl of PI. Cells were incubated at room temperature for 15 min in the dark and fluorescence was analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA). At least 10,000 events were recorded. Apoptotic cells were expressed as a percentage of the total number of cells. Apoptosis of cardiomyocytes were analyzed with an in situ cell death detection kit as described in our previous publication [12 (link)]. The staining was observed under a Zeiss confocal laser scanning microscope (Carl Zeiss, LSM 780 Meta Confocal Laser Scanning Microscope). The experiment was repeated three times.