TUNEL Assay

Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to detect apoptosis-specific nuclear DNA fragmentation. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature before TUNEL staining. The fixed cells were then stained using a commercial TUNEL kit (In Situ Cell Death Detection Kit; Boster, Wuhan, China) according to the manufacturer’s recommended instructions. TUNEL-positive cell nuclei were visualized based on green fluorescence and imaging was performed under 40× magnification. The percentage of TUNEL-positive cells was calculated in five microscopic fields from each slide. The population of TUNEL-positive cells was also quantified by flow cytometry.