Isolation and culture of mouse primary hepatocytes

Primary hepatocytes were isolated from 8- to 12-week-old male C57BL/6J mice as described previously (Sakai et al., 2012). Mice were humanely euthanized by exposure to CO₂. Livers were perfused in a retrograde fashion for 3 minutes at a rate of 5 ml/min through the inferior vena cava with HBSS with Ca or Mg (Gibco) supplemented with 10 mM HEPES (Gibco), then for 18 minutes with the same solution containing collagenase type I (32 mg per 100 ml, Worthington) and Protease Inhibitor Cocktail Complete–EDTA Free (Roche). The hepatocytes were harvested and purified by density gradient centrifugation with Percoll (Sigma) and plated on type I collagen–coated six-well plates (1 million cells per well) in Medium 199 (Gibco) supplemented with 5% FBS and 100 U/ml penicillin/streptomycin (Thermo Fisher Scientific).