Methyl thiazolyl tetrazolium (mtt)

To evaluate the cell viability, MC3T3-E1 cells were stimulated with Al₂O₃, Ti, and UHMWPE particles at various doses (0, 25, 50, 100, 200, 400, 800, and 1000 μg/mL) for 24 h, and then 5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide was added (MTT; Sigma Aldrich, St. Louis, MO, USA). The supernatant was removed after incubating with MTT at 37 °C for 2 h. Next, 200 μL of DMSO was added to each well, and we measured the optical density at 570 nm using a UV-Vis spectrophotometer (SpectraMax, Molecular Devices, San Jose, CA, USA).

Cells (2000–3500 cells per well depending on the cell line) were cultured overnight in flat-bottomed 96-well plates. Cell viability was assessed after the addition of sulforaphane at the indicated concentration or vehicle control (0.05% DMSO) for 72 h. At the end of the treatment, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Biovision, Milpitas, CA, USA) was added and the plates were incubated for a further 4 h. During the 72 h cell viability assay, the medium was not changed. The number of viable cells was estimated by the formation of formazan product as a result of conversion of MTT by viable cells using a VersaMax enzyme-linked immunoassay microplate reader (Molecular Devices, Sunnyvale, CA, USA). To evaluate whether inhibitors of specific pathways prevented sulforaphane-induced cell death, cells were incubated with the inhibitors for 1 h before treatment with sulforaphane.

For MTT assays, 3000–15000 cells were seeded in 80 μl of media. Next day 20 μl (5 times concentrated) of several drug concentrations prepared in 1:2 serial dilutions were added in triplicates. After 72 h, 10 μl of MTT (5 mg/ml) (Sigma) was added and incubated for 3 h. Thereafter, 50 μl of SDS lysis buffer (10% SDS ^w/_v, 0.01 M HCl, pH 5.5) was added and incubated overnight. MTT absorbance was read using SpectraMax 190 plate-reader (Molecular Devices) at 570 nm. 650 nm was used as a reference wavelength. Percentage survival for each dose was calculated by multiplying absorbance values by 100 and dividing by control absorbance value. Dose-response curves were drawn using GraphPad Prism which were used to determine IC₅₀ values.
For clonogenic assays, 1000 cells were seeded in 6 well plates. After two days compounds were added. Media and compounds were replenished every four days. The plates were incubated until several colonies appeared. For colony staining, media was aspirated followed by PBS wash. Colonies were fixed by buffered neutral 10% formalin (Sigma) for 2 h and washed with PBS three times. Methylene blue (0.3% (w/v), Sigma) was added to wells and incubated overnight. Plates were then washed several times with water to remove excessive stain and photographed.

Cell proliferation was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Aldrich, Milan, Italy). PC-3, DU-145, and LNCaP cancer cells (10³ cells/well) were seeded into a 96-well plate. Following cell adhesion, fresh complete medium with decreasing dilutions (ranging from 1:50 to 1:10,000) of A009 or HyT were added for 24 to 96 h. EtOH dissolved in the RPMI complete medium was used as control vehicle. MTT reagent 5 (mg/mL) was added to cell cultures, which was then replaced with 100 μL of DMSO, and the amount of solubilized formazan was quantified at 570 nm in a SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA).