Primary Hepatocyte isolation and culture

Hepatocytes were isolated from 8-week-old C57Bl/6 or C57Bl/6 NOX1^−/− mice according to previously described methods [17]. Briefly, mice were anesthetized with isoflurane and the portal vein was exposed and cannulated with a 26G catheter. The liver was perfused and digested with collagenase type IV (Sigma). Hepatocytes were then purified via Percoll centrifugation and seeded at a density of 1.5 × 10⁵ hepatocytes per well onto 48-well plates coated with 0.17 mg/ml rat tail Collagen-1 (BD Biosciences). Hepatocytes were cultured in media containing DMEM with high glucose (4.5 g/L), 10% (v/v) fetal bovine serum (Biowest), 0.04 µg/ml dexamethasone, 7 ng/ml glucagon, 1% ITS+culture supplement (Corning), 1.5% 1 M HEPES, and 1% penicillin-streptomycin. The next day, hepatocytes were infected. Replicate experiments were performed with both male and female mice.