Glutamine assay kit wst

Cells (2.5 × 10⁵) were seeded on 100-mm culture plates overnight and allowed to grow to the logarithmic stage with 0, 1, and 10 mM metformin in complete medium. Treatment medium was changed every 24 h to maintain a stable nutrient supply. Culture media were collected after 72 h for HSC-2, HSC-3, and HeLa and after 96 h for HaCaT, and nutrient consumption was evaluated. Glucose concentrations were assessed using the Glucose C2 test (134-19121, Fujifilm-Wako Pure Medical Corporation, Miyazaki, Japan) and glutamine concentrations by the Glutamine Assay Kit-WST (G268, Dojindo, Kumamoto, Japan) according to the manufacturers’ instructions. Pyruvate concentrations were evaluated by HPLC.

ADSM were pre-cultured for 16 h in DMEM (Wako, High glucose) without FBS before CM was added directly to a final concentration of 50% and the cells were cultured for another 24 h. The glucose and glutamine concentrations in the medium were measured using a Glucose Assay Kit-WST (DOJINDO, Japan) and Glutamine Assay Kit-WST (DOJINDO), respectively. Absorbance at 450 nm was measured using a FluxStation3 (Molecular Devices) according to the manufacturer’s instructions.