2.4. TUNEL Assay

Apoptosis in H9c2 cells was detected by using a high-sensitive one-step terminal deoxyribonucleotide transferase dUTP nick end labelling (TUNEL) apoptosis assay kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's instructions. In brief, H9c2 cells were fixed with 4% paraformaldehyde for 30 min. After washing with PBS, using 0.3% Triton X-100, H9c2 cells were permeabilized. Then they were incubated with 50 μL TUNEL reaction fluid in a humid environment at 37°C for 1 h. And after washing twice with PBS, H9c2 cells were incubated with 4′, 6-diamidino-2-phenylindole (DAPI) to stain nuclei. Finally, H9c2 cells were observed under fluorescence microscope.