KCs were isolated using the method described by Yue et al.20 (link) Briefly, the portal vein was exposed, a surgical thread was inserted in the portal vein and the liver was perfused with HBSS solution prewarmed at 37°C. The perfusion solution was then switched to type IV collagenase for 20 m, until the liver had slightly swelled. The liver was removed and washed with phosphate-buffered saline for three times, and then the liver was finely chopped and digested with collagenase solution for 20 m by vigorous shaking at 37°C. The resultant cell suspension was teased through a cell strainer (100 µm) and the hepatocytes in the supernatant was removed by three 2 m centrifugations at 50×g. Then the nonparenchymal cells (NPCs) were layered onto a 50%/25% two-step Percoll gradient (Sigma) and centrifuged at 2,000×g for 10 m at 4°C. KCs in the middle layer were collected.