100mm tissue culture dishes

For the assessment of the cell death in PC3, cells were cultured in 100 mm tissue culture dishes (Fisher Scientific, Hampton, NH) using RPMI phenol red free medium and reach a growth ~75% confluency. The cells were dose using the IC₅₀ of compounds a, 1a, 2a, AF and incubated for 72 h at 37 °C under 5% CO₂ and 95% air in a humidified incubator. Then, cells were collected using trypsin (Fisher Scientific, Hampton, NH) to later count with a hemocytometer and prepare 25 ×104 cells/sample. After the samples were prepared, the cells in the samples were centrifuged, supernatant was aspirated from the pallet, and washed gently one time with PBS. The cells were centrifuged again, PBS was aspirated and eBioscience Annexin V-FITC Apop Kit (Invitrogen, Carlsbad, CA) was used to labeled cells as follows, the cells were resuspended in 195 μL of 1x binding buffer and 5 μL of Annexin-V dye was added. The cells were incubated at room temperature for 10 min. After the incubation period finished, cells were washed one time with 1x binding buffer and resuspended in 190 μL of 1x binding buffer with 10 μL of propidium iodide. The dye’s fluorescence intensity was detected via flow cytometry using a BD C6 Accuri flow cytometer. 10 × 105 events per sample were recorded. The flow cytometer was calibrated prior to each use.