Measurement of Cytosolic and Mitochondrial ROS in HepG2 Cells

Cytosolic and mitochondrial ROS were measured on a 96-well plate reader as previously described (28 (link)). Briefly, the HepG2 cells were seeded at a concentration of 1×10⁴ cells per well in black 96-well flat bottom plates (Thermo Fisher Scientific, Inc.) and allowed to adhere overnight. Following seeding, the cells were washed and incubated with serum-starved medium, D-GalN (50 nM), 5% hPH, or 5% hPH + D-GalN (50 mM) for 4 h, followed by incubation with 2′,7′-dichlorofluorescin diacetate (DCFH-DA; 10 µM, ex/em: 518/605 nm; Invitrogen; Thermo Fisher Scientific, Inc.) or MitoSOX™ (5 µM, ex/em: 510/580 nm; Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min. The oxidative products were measured with the SpectraMax i3x Multi-Mode detection platform.