Wst 1 viability assay

CCa cells (2.0 × 10⁴/well) were seeded in a 96-well plate. 24 h later, the culture medium was removed and cells were treated with a range of concentrations of oxaliplatin, paclitaxel, and vincristine as indicated for another 48 h. Subsequent cell viability was determined using the WST-1 viability assay (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol. The optical density was measured at 450 nm with 650 nm as the reference wavelength with a microplate reader (xMark™ Microplate, Bio-Rad, Hercules, CA, USA). Based on the drug response curves, we determined the IC50 values (drug concentrations that achieved 50% growth inhibition) in respect to the different drug treatments using Bliss method [21 (link)]. Evaluation of cell apoptosis was carried out using an apoptosis ELISA kit (Roche Diagnostics, Shanghai, China), as per the manufacturer's instructions.

D11-mediated cytotoxicity was determined by the WST-1 viability assay (Roche, Hvidovre, Denmark). Viability was quantified in a microtiter plate reader (VersaMax ELISA, Molecular Devices, Sunnyvale, CA, USA) after adding the WST-1 reagent to the cells according to the manufacturer’s guidelines.

SKOV3 and OVCAR3 cells were treated with vehicle or ML246 for 72 h at the indicated concentrations. Cell viability was measured using the WST-1 viability assay as per the manufacturer’s instructions (Roche, Switzerland).
In vitro invasion assays were performed on SKOV3 and OVCAR3 using 24-well transwell units with polycarbonate filters (pore size: 8 μm) coated on the upper side with reconstituted basement membrane matrix, Matrigel (BD Biosciences, USA). 5 × 10⁵ cells were added to the transwell in serum free media. Media with FBS was added to the outer compartment as the chemoattractant. Cells were treated with DMSO or ML246, and cultured for 72 h at 37 °C. Cells remaining on the upper side of the transwell were scraped off with a cotton swab. Cells remaining on the underside of the membrane were fixed and stained using the commercially available, Shandon Kwik-Diff stain kit (ThermoFisher Scientific). Cells were counted in four fields and the mean + SEM was calculated. This experiment was performed in triplicate in three independent experiments.