Apoptosis Assay for PC-3 and DU145 Cells

Late apoptotic cell death was determined using a Roche Diagnosis TUNEL assay kit. PC-3 (5 × 10⁵ cells/well) and DU145 cells (5 × 10⁵ cells/well) were treated with EVO with or without HGF for the indicated times and at the indicated concentrations and then were fixed in 4% paraformaldehyde at room temperature for 30 min, washed with 1× PBS, and permeabilized with 0.2% Triton X-100 at room temperature for 15 min. The cells were then washed with 1× PBS and resuspended in TUNEL reaction mixture for 1 h at 37 °C in the dark. DNA damage in the stained cells was analyzed by a BD Accuri™ C6 Plus Flow Cytometer (BD Biosciences, Becton-Dickinson, Franklin Lakes, NJ, USA) using the BD Accuri C6 Plus software [48 (link)].