Quantichrom ethanol assay kit

Ethanol or acetaldehyde were added to FW, EHW (LV4), AC, or HW to prepare 4% ethanol and 1 mM acetaldehyde solutions in 50 mL tube, respectively. The solutions were stored at room temperature for 24 h. Then, the ethanol concentration was measured using the QuantiChrom™ Ethanol Assay Kit (BioAssay Systems, DIET-500), according to the manufacturer’s protocol. Briefly, 100 µL of these waters that were mixed with 4% ethanol and standard were added to each well at 96-well plate, followed by addition of 100 µL of Reagent A, colorimetric assay buffer. After incubation at room temperature for 8 min, the absorbance was determined by measuring at 570 nm with Multiskan™ FC (Thermo Scientific™, MA, USA). Additionally, the acetaldehyde concentration was measured using the EnzyChrom™ Acetaldehyde Assay Kit (BioAssay Systems, EACT-100) according to the manufacturer’s protocol. Briefly, 20 µL of these waters that were mixed with 1 mM acetaldehyde and standard were added to each well at 96-well plate, followed by addition of 80 µL of Warking Reagent, including Assay buffer, NAD/MTT solution, Enzyme A, and Enzyme B. After incubation at room temperature for 30 min, the absorbance was determined by measuring at 570 nm with Multiskan™ FC.

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 treated for 24 h with medium that was mixed with 4% ethanol and FW, EHW (LV4), AC, or HW. Ethanol concentration in the culture medium was measured using the QuantiChrom™ Ethanol Assay Kit (BioAssay Systems, DIET-500) according to the manufacturer’s instruction. Briefly, the culture medium was deproteinated by adding 1 volume of these medium to 2 volumes of 10% TCA, followed by centrifuge at 14,000 rpm for 5 min, and their supernatant was used for the assay. Colorimetric quantification was performed in the same way as in Section 2.5.