Figure 7: In caspase assay, PC3 cells (1 × 105 cells/mL) were treated with subtoxic concentration (10 μg/mL) of B2. Betulinic acid (10 μg/mL) and DMSO (0.1%) were used as positive and negative controls. The cells were labeled with fluorescent FAM-VAD-FMK dye after 8 hours of treatment and incubated for 60 minutes. The media were removed thereafter and the cells were incubated in fresh media for another 60 minutes. The plate was centrifuged at 300 RPM for 5 minutes to include the detached apoptotic cells in the assay. Then the media were replaced by PBS in each well. The fluorescence intensity was recorded at 490 nm excitation and 520 nm emission using a fluorescence microplate reader (Multiskan Ascent micro plate reader, Thermolab system 354, Finland). The apoptotic cells were observed under fluorescent microscope at 20x magnification. (a) Photomicrograph of the fluorescence emitting cells. The images were taken by EVOS fluorescence microscope at 20x magnification. (b) Graphical representation depicts the quantitative estimation of percent induction of caspase activity in PC3 cells by B2 and betulinic acid (BA).