Alcohol dehydrogenase activity colorimetric assay kit

HepG2 cells were seeded in a 6-well plate at a density of 2.0 × 10⁵ cells/well for 24 h. Then, the cells were incubated for 24 h with medium that was mixed with 4% ethanol and FW, EHW (LV4), AC, or HW. The cellular ADH activity was measured using the Alcohol Dehydrogenase Activity Colorimetric Assay Kit (BioVision, K787-100) according to the manufacturer’s instruction. Briefly, the cells were lysed with ADH Assay Buffer, 50 µL of which were mixed with 100 µL of Reaction Mix, including ADH Assay Buffer, Developer, and Substrate, to each well at 96-well plate. The mix was incubated for 3 min at 37 °C, then the absorbance was measured at 450 nm with Multiskan™ FC, which was expressed as Abs_0min, and was incubated for another 60 min at 37 °C and the absorbance was measured at 450 nm again, which was expressed as Abs_60min. The ∆Abs (= Abs_60min − Abs_0min) was applied to the NADH standard curve to get X nmol of NADH generated by ADH during the reaction time (60 min). The ADH activity was calculated from the following equation:

Then, the cell lysate volume was converted to the number of cells and counted by trypan blue method, and the ADH activity was recalculated (µU/1.0 × 10⁶ cells).