Isolation and Culture of Primary Hepatic Cells

Primary hepatocytes and Kupffer cells from the SMO^FL/FL, SMO^M-KO, or wild-type (WT) mice were isolated as described (22 (link)). In brief, the livers were perfused in situ with warmed (37°C) HBSS solution, followed by a collagenase-buffer (collagenase type IV, Sigma-Aldrich). Perfused livers were dissected and teased through 70-μm nylon mesh cell strainers (BD Biosciences). Nonparenchymal cells (NPCs) were separated from hepatocytes by centrifuging at 50 × g for 2min three times. NPCs were suspended in HBSS and layered onto a 50%/25% two-step Percoll gradient (Sigma) in a 50-ml conical centrifuge tube and centrifuged at 1800 × g at 4°C for 15min. Kupffer cells in the middle layer were collected and allowed to attach onto cell culture plates in DMEM for 15min at 37°C. Bone marrow-derived MSCs were isolated, as described (23 (link)). In brief, the bone marrow cells were cultured with α-MEM. After 24h, the culture medium was refreshed to remove non-adherent cells. MSCs were used in the experiments only after 2 to 3 expansion passages to ensure depletion of monocytes/macrophages. Murine bone-derived macrophages (BMMs) were generated as described (22 (link)). See Supplementary Materials.