TdT-mediated dUTP-biotin Nick end-labeling (TUNEL) Staining
The TUNEL assay was performed using DeadEnd Fluorometric TUNEL System kit (Promega, Madison, WI, USA). In brief, cells were fixed with 4% paraformaldehyde at 4°C for 25 min, penetrated with 0.2% Triton X-100 for 5 min, and incubated with 100 μL of equilibration solution per well at room temperature for 5 - 10 min. After aspirating the equilibration solution, 50 μL of the terminal deoxyribonuclease reaction solution containing 45 μL of equilibration buffer, 5 μL of nucleotide mixture, and 1 μL of terminal deoxynucleotidyl transferase was added to each well, and the reaction was carried out at 37°C for 1 h. Then each well was mixed with 50 μL of 2 × soluble solids content (SSC) at room temperature for 15 min in the dark to terminate the reaction. After washing three times in PBS, cells were stained with 4’,6-diamidino-2-phenyl indole (DAPI) (1 μL/mL) at room temperature for 15 min, followed by rinsed with PBS for three times. Next, cells were subjected with anti-fade solution (Molecular Probe, Cat#57461) and photographed under fluorescence microscope (IX71-F22FL/DIC, Olympus America Inc., Bethlehem, USA). Five visual fields of each group were randomly selected for observation, and the number of cells was counted.