2.5. The Cell Culture and Treatment

The murine macrophage cell line RAW264.7 was bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The murine normal hepatic cell line NCTC1469 was obtained from China Center for Type Culture Collection (Wuhan, China). Both cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose) with 10% fetal bovine serum under a humidified atmosphere of 5% CO₂ at 37°C. RAW264.7 cells (2∗10⁵ per well) were seeded in 6-well plates overnight. Then, OI (100 μM) or vehicle was added 2 h before LPS (200 μg/ml) administration. Then, cells were collected for western blot or immunofluorescence 6 h later. For measurement of cell viability, NCTC 1469 cells (1∗10⁴ per well) were seeded in 96-well plates overnight. Different concentrations of D-GalN (10 mM, 20 mM, 30 mM, and 40 mM) or OI (10 μM, 50 μM, and 100 μM) were added, respectively, for 12 h to determine a suitable concentration for the next experiments. Then, NCTC 1469 cells (2∗10⁵ per well) were seeded in 6-well plates overnight. OI (100 μM) was added 2 h before D-GalN (40 mM) treatment. After D-GalN was given for 12 h, cells were collected for western blot, ROS assay, and Annexin V/7-amino-actinomycin D staining.