Af 300 01a

Primary hepatocytes were isolated from C57BL/6J mice and cultured as described in a previous study.⁴¹ (link) Briefly, mice were deeply anesthetized with isoflurane and perfused with digestion buffer containing 100 CDU/mL (CDU = collagen digestion units) type IV collagenase through the inferior vena cava. Once the digestion was done, the liver cells were extracted and filtered through a 70-μm membrane. After centrifugation, the supernatant containing nonhepatocytes and the remaining hepatocyte pellet were transferred, washed 3 times, and collected separately. For the treatment of hepatocytes, the isolated primary hepatocytes continued to be plated into 12-well culture dishes coated with collagen at 2.8 × 10⁵ cells/well. Hepatocytes were cultured in MEMα (Minimum Essential Medium α) with 10% fetal bovine serum (Gibco, Waltham, MA) plus 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Waltham, MA) at 37°C with a humidified atmosphere of 5% CO₂ for 4 hours, and then kept in serum-free medium until the primary hepatocytes were treated with serum-free medium containing 0.75 mmol/L PA (P0500; Sigma, Darmstadt, Germany), 100 ng/mL lipopolysaccharide (L4391; Sigma, Darmstadt, Germany), and 20 ng/mL recombinant tumor necrosis factor α (AF-300-01A; Peprotech, Cranbury, NJ) for 24 hours.