4.8. TUNEL Assay

Late apoptotic cell death was ascertained with a TUNEL (terminal transferase mediated dUTP-fluorescein nick end labeling) assay kit (Roche Diagnostics GmbH, Penzberg, Germany), in accordance with the instructions from the manufacturer. In brief, U266 cells were treated with 100 µM of DHAP for 24 and 48 h, then washed with cold PBS. The cells were seeded after being fixed with 4% paraformaldehyde for 30 min and washed twice with PBS. Following this, the resuspended cells were placed in a permeabilization solution (0.1% Sodium citrate and 0.1% Triton X-100) for 20 min at 4 °C, then washed with cold PBS. The cells were subsequently incubated with a TUNEL label mixture and TUNEL enzyme for 1 h at 37 °C in darkness. Finally, the cells were washed with PBS and analyzed using Cell Quest 3.0 software with a flow cytometer.