Isolation and treatment of hepatic stellate cells

As described previously^{5 (link),23 (link)}, hepatic stellate cells (HSCs) were isolated by in situ collagenase perfusion of the liver. After anesthesia by intraperitoneal injection with 100–200 mg/kg ketamine/xylazine cocktails, the liver was perfused in situ with collagenase type I through the portal vein. Non-parenchymal cells were separated by centrifugation at 500 rpm and 4 °C for 5 minutes. Then, the HSCs and Kupffer cells were isolated by differential centrifugation on a density gradient of 11.5% and 20% OptiPrep (Sigma-Aldrich, MO, USA) at 3,000 rpm and 4 °C for 17 minutes. HSCs and Kupffer cells were located in the upper and lower layers, respectively. Kupffer cells were positively purified by labeling with anti-mouse F4/80 microbeads (Miltenyi Biotec) and subjected to real-time PCR analysis. HSCs and Kupffer cells were treated with alcohol (50–200 mM), lipopolysaccharide (10 ng/mL; Sigma-Aldrich, MO, USA), or recombinant GDF15 (100 ng/mL; R&D Systems, Minneapolis, MN, USA).