2.7. Measurement of reactive oxygen species (ROS)

The primary hepatocytes were pre-loaded with the ROS probe (CM-H2DCFDA, Thermo Fisher Scientific) at 50 μg/24-well plates for 30 min at 37 °C. Then hepatocytes were treated with ethanol (100 mM) and NAC (2 mM) or H₂O₂ (10 μM) for an additional 60 min at 37 °C. Data were collected using a plate reader (BioTek Synergy H1) with excitation at 492 nm and emission at 522 nm.