Quantifying Apoptosis via TUNEL Assay

In order to detect apoptosis, TUNEL assay was used as previously described25 (link). Briefly, cells were fixed with 4% paraformaldehyde solution for 0.5 h at room temperature. Then the cells were exposed to a methanol solution containing 0.2% H₂O₂ for 0.5 h to block endogenous peroxidase activity. TUNEL reaction mixture (Sigma-Aldrich) was then added, and the cells were placed in an incubator at 37°C for 60 min. Finally, laser confocal microscopy (A1; Nikon, Japan) was used to capture the fluorescence. Apoptosis index was quantified as the ratio of (TUNEL⁺ cells)/(total cells) × 100%.