*3.5. Cytotoxicity Assay*

Human prostate adenocarcinoma cells (PC3) and human neonatal foreskin fibroblast (noncancer cells, NFF) were grown in media RPMI-1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS) (Life Technologies, Grand Island, NY, USA). Cells were grown under 5% CO2 in a humidified environment at 37 ◦C. Fifty microliters of media containing 500 cells were added to a 384 well microtiter plate (Perkin Elmer, Shelton, CT, USA, part number: 6,007,660) containing 0.2 μL of a compound. Final compound concentrations tested were 20, 6.5, 2, 0.65, 0.2, 0.065, 0.020 and 0.0065 μM (final DMSO concentration of 0.4%). Each concentration in media was tested in triplicate and in two independent experiments. Cells and compounds were then incubated in 72 h at 37 ◦C, 5% CO2 and 80% humidity. Cell proliferation was measured with the addition of 10 μL of a 60% Alamar blue (Invitrogen, Carlsbad, CA, USA) solution in media to each well of the microtiter plate to give a final concentration of 10% Alamar blue. The plates were incubated at 37 ◦C, 5% CO2 and 80% humidity within 24 h. The fluorescence of each well was read at excitation 535 nm and emission 590 nm on the Perkin Elmer EnVision Multilabel Reader 2104. Eight-point concentration response curves were then analyzed using non-linear regression and IC50 values determined by using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Paclitaxel and doxorubicin were used during each screening as positive control compounds.
