4.8. Apoptosis by ELISA

LNCaP and DU145 cells were treated with 20 µM BA in the presence of pan caspase inhibitors Z-VAD-FMK and the caspase 3 inhibitor, DEVD-CHO, for 48 h and ELISA for apoptosis was performed using the Cell Death Detection ELISA^PLUS kit following the manufacture’s protocol. Briefly, the cells after 48 h treatment with BA the cells were washed with cold PBS and lysed using Tris-HCl lysis buffer for 30 min. The lysate was clarified by centrifugation at 5000 rpm for 10 min at 4 °C and the supernatant was stored at −80 °C. Then, 50 μg of cell lysate protein were added to lysis buffer provided with the kit and pipetted on a streptavidin-coated 96-well microtiter plate to which immunoreagent mix was added and incubated for 2 h at room temperature with continuous shaking at 500 rpm. The wells were then washed with washing buffer, and color was developed by addition of substrate solution, which was read at 405 nm against the blank reference wavelength of 490 nm after 10–15 min. The enrichment factor (total amount of apoptosis) was calculated by dividing the absorbance of the sample (405 nm) by the absorbance of the controls without treatment (490 nm).