TUNEL assay

Cell apoptosis was determined by TUNEL assay, conducted using DeadEnd Fluorometric TUNEL System (Promega) and performed according to the manufacturer’s instructions. In brief, cells were treated for 18 h, as indicated, then were fixed in freshly prepared 4% paraformaldehyde solution in PBS (pH 7.4) for 25 min at 4 °C. After fixation, cells were permeabilized in 0.2% Triton X-100 solution in PBS for 5 min. After washing twice with washing buffer for 5 min, the cells were covered with equilibration buffer at room temperature for 5–10 min. The labeling reaction was performed using terminal deoxynucleotidyl transferase end-labeling TdT and fluorescein-dUTP cocktail for each sample and incubated for 1 h at 37 °C where TdT catalyses the binding of fluorescein-dUTP to free 3’OH ends in the nicked DNA. After rinsing, cells were washed with 2× SSC solution buffer and subsequently incubated with propidium iodide (Sigma-Aldrich) to stain nuclei and analyzed using the Cytation 3 Cell Imaging Multimode Reader (BioTek, Winooski, VT, USA).