Isolation and Purification of Hepatic Leukocytes and Hepatocytes

Hepatic leukocytes were isolated as described previously [24 (link)]. Briefly, liver tissues were minced and sieved through a 70 μm filter. Hepatic leukocytes were purified by centrifugation on a 40% Percoll gradient (GE Healthcare), and red blood cells were lysed by RBC lysis buffer. Collected leukocytes were further enriched using PE-anti-F4/80 (BD Pharmingen, Cat# 565410) and anti-PE microbeads (Miltenyi Biotec) to isolate macrophages. Hepatocytes were isolated as described previously [25 (link)]. Briefly, the liver was perfused with a solution of EGTA and digested with a 0.075% collagenase solution. The viable hepatocytes were separated by 40% Percoll solution with centrifugation at 420 × g for 10 min at 4 °C.