Intracellular ROS Measurement in H2O2-induced HepG2 Cells

The measurement of ROS in H₂O₂-induced HepG2 cells was carried out according to the method described by Zhao with minor modifications [18 (link)]]. HepG2 cells were grown for 12 h with a density of 1 × 10⁵ cells/mL in a 6-well plate. The sample groups (100 μg/mL of 5 DH fractions, 1 mL), the VC group (100 μg/mL VC, 1 mL), and the H₂O₂ group (1 mL DMEM) were mixed with 1.0 mmol/L H₂O₂ for 24 h, while the control group was treated only with 2 mL DMEM solution for 24 h. After digestion by trypsin, 1 mL of PBS was added to the cells for the collection of cell supernatants. The cell supernatants were then incubated with 1 mL of DCFH-DA (10 μM) at 37 °C for 30 min in the dark. The intracellular green fluorescence intensity was measured at 485 nm excitation and 525 nm emission wavelengths by flow cytometry (Guava^® easyCyte 6-2L, Millipore, Billerica, MA, USA).