Isolation and Culture of Primary Mouse Hepatocytes

Primary mouse hepatocytes (PMoH) were isolated from up to 12-week-old C57BL/6 mice, as previously described[12 (link)]. In brief, PMoH were extracted via ex-vivo perfusion of the left liver lobe with Ca²⁺ and Mg²⁺ free HEPES buffer (Invitrogen), followed by HEPES containing 500 mg/l collagenase IV (Sigma-Aldrich). Hepatocytes were separated on a 45% Percoll density gradient (Sigma-Aldrich) and seeded at a density of 50,000 cells/cm² in cell culture plates coated with 0.6 mg/ml rat-tail collagen (Sigma-Aldrich). PMoH were cultured overnight in complete Williams E medium (Invitrogen) containing 10% FBS, 1% glutamine (Sigma-Aldrich), 50μg/ml penicillin/streptomycin (Invitrogen) and 1:1000 gentamicin (Invitrogen). Prior to infection with recombinant adenoviruses, non-adherent cells were washed off and adherent cells incubated with complete Williams E medium supplemented with 1% HEPES pH 7.4 (Invitrogen), 0.1% gentamicin (Invitrogen), 1% glutamine (Invitrogen), 1% linoleic acid (Sigma-Aldrich), 1% epidermal growth factor (EGF) (BD BioScience), 0.1% ITS (Sigma-Aldrich), 0.1% insulin (Sigma-Aldrich), 0.01% dexamethasone (Sigma-Aldrich) and 0.01% ethanolamine (Sigma-Aldrich).