2.3. Isolation of primary hepatocytes of mice

Primary hepatocytes were isolated from male mice at 8–12 weeks of age as previously described [21]. Briefly, collagenase perfusion was performed with 50 ml of perfusion buffer through the portal vein of mice after anesthetizing. Livers were aseptically removed and minced in a sterile 10-cm cell culture dish with ice-cold perfusion buffer without collagenase. Then, hepatocytes were filtrated through a 70 µm cell strainer (BD Falcon) into a new tube followed with centrifugation at 50 g for 2 min at 4 °C. Cells were then washed with cold hepatocyte wash medium (Invitrogen) three times and re-suspended in 15 ml of cold HepatoZYME-SFM medium (Invitrogen) supplemented with 2 mM L-glutamine, 20 units/ml penicillin, and 20 μg/ml streptomycin. After trypan blue staining for determination of viability, cells were plated at 6 × 10⁵ cells/well in 6-well culture dishes or at 3 × 10⁵cells/well in 12-well dishes pre-coated with collagen. Cells were cultured for at least 8 h before further use. Cells were subsequently treated with the desired reagents prior to further analysis.