Cell Viability Assay with Metabolic Modulators

HT29 and HCT116 cells were cultured in 96-well plates (2 × 10⁴ cells/well) and maintained for 24 h. Then, the cell culture medium was replaced with McCoy’s 5A medium without serum and incubated at 37 °C with 5% CO₂ in a humidified atmosphere for 12 h. Subsequently, the cells were treated with 50 mM glucose (Wako, Osaka, Japan), 100 nM recombinant human insulin (Novo Nordisk Pharma, Tokyo, Japan), 250 µM OA (Sigma-Aldrich, Germany), 250 µM PA (Sigma-Aldrich, Germany)^{45 (link)}, 100 µM AICAR (AdipoGen, CA, USA), and phosphate-buffered saline as control (Ctrl) for 6 h, or siRNA for 72 h. After treatment, 10 µL of Premix WST-1 solution (TaKaRa, Japan) was added to each well and incubated for 6 h at 37 °C. After incubation, the absorbance was measured at 450 nm using a microplate reader (Benchmark Plus Microplate Reader, Bio-Rad, USA), at a wavelength of 690 nm.