ELISA apoptosis assay

For the determination of the apoptosis assay by histone-associated fragmented DNA, 2 × 10⁴ PC3 live cells were seeded per well (200 μL volume) in a 96-well plate and were treated under the same conditions described above for 24 h at 37 °C, 95% air, and 5% CO₂. The cell plates were centrifuged at 1200 rpm for 10 min at 4 °C. Supernatants were decanted and the cells were lysed for 30 min in 200 μL of lysis buffer, centrifuged at 200 × g for 10 min. Twenty μL of lysate of each sample was transferred onto the Streptavidin-coated microplate plus 80 μL immunoreagent per well. The samples were incubated for 30 min and were protected from light at between 15 and 25 °C. The cells were centrifuged at 1200 rpm for 10 min at 4 °C, and 20 μL of the supernatant from each well was taken and placed into the ELISA 96-well kit plate. Eighty μL of immunoreactive was added to each well (Incubation Buffer 72 μl, Anti-Histone 4 μl, Anti-DNA 4 µL), the plates were covered with an adhesive cover, and these were incubated in a shaker at 300 rpm for 2 h at between 15 and 25 °C. The plates with the supernatants were removed. Each plate well was washed 3 times with 300 µL of incubation buffer; 100 μL of ABTS solution was added to each well. The plates were incubated in a shaker at 250 rpm for 20 min; 100 μL of ABTS stop solution was added to each well. The absorbance of each sample was determined using a microplate reader (Sinregy HT Multi-Mode Microplate  Reader, Biotek at 450 nm). In the DNA fragmentation test, the rate of apoptosis is reflected by the Enhanced factor (fold change) of mono- and oligonucleosomes accumulated in the cytoplasm, both of which were calculated and normalized versus UCG.