DNA fragmentation assay

Cell death was determined by measuring fragmented DNA using Cell Death Detection enzyme-linked immunosorbent assay (ELISA) kit (Roche Applied Science, Mannheim, Germany). Briefly, cells were lysed by adding cell lysis buffer supplied with the kit. After centrifugation (200 × g, 10 min), the supernatant was pipetted onto an anti-streptavidin-coated microplate. Anti-DNA monoclonal antibody conjugated with peroxidase (anti-DNA-POD) and anti-histone-biotin were added. After incubating at 25°C for 90 min, wells were rinsed with incubation buffer (supplied by the kit) three times. The color was developed by adding 2,20-azino-di-[3-ethylbenzthiazoline sulfonate] (ABTS) substrate solution and incubated at room temperature for 10−20 min with shaking (250 rpm). The amount of peroxidase retained in the nucleosome complex was determined by measuring absorbance at a wavelength of 405 nm.