*2.5. Isolation of Hepatic Stellate Cells*

HSCs were isolated from 4–6 month-old female WT C57BL/6 mice. Mice were kept under the same conditions as described above. Mice were anesthetized and placed under a heating lamp. After cannulation of the portal vein, the liver was perfused using HBSS buffer (37 ◦C). Following a perfusion with pronase E (0.2% in HBSS buffer) and collagenase type I (0.025% in HBSS buffer), that led to digestion of collagen and lysis of hepatocytes, the liver was removed and mechanically diced. The liver cell solution was filtered and resuspended in a pronase/Dnase solution (0.125%) for further digestion. After filtration through a 70 μm cell strainer, the solution was diluted in HBSS and centrifuged at 500× *g* for 7 min. The supernatant containing cell debris was removed. The cell pellet was resuspended in HBSS containing 0.25% BSA and then mixed with 28.7% Nycodenz gradient solution (Cosmo bio USA, Carlsbad, CA, USA) to obtain a final concentration of 18% Nycodenz (*w*/*v*). This preparation was covered with 8 mL 0.25% BSA/HBSS before centrifugation for 30 min at 1400× *g* without brake. After centrifugation, the layer between the two buffers was harvested and centrifuged at 450× *g* for 10 min. The supernatant was discarded and the pellet was resuspended in DMEM supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin/amphotericin B. The medium was exchanged every other day. Cells were kept on cell culture plates covered with collagen type 1 in a density of 2.5–3 <sup>×</sup> <sup>10</sup><sup>6</sup> cells/well in a 6-well plate. Cells were used for experiments on the next day or after seven days for experiments with activated HSCs (Supplemental Figure S1). During experiments, cells were kept in DMEM without FCS or antibiotics.
