Anti mouse f4 80 microbeads

NSE-BMP4 mice were sacrificed, and the hindlimbs were harvested to isolate the long bones (including the femur and tibia) in a biosafety hood. PBS injected with a syringe and needle was used to flush out the bone marrow from the femur and tibia. Red blood cells in the flushed bone marrow were lysed with RBC lysis buffer (NH₄Cl (8.99 g·L⁻¹) + KHCO₃ (1 g·L⁻¹) + Na₄-EDTA (0.037 g·L⁻¹)). F4/80⁺ macrophages were sorted using a MACS kit containing anti-mouse F4/80 microbeads (130-110-443, Miltenyi) according to the manufacturer’s instructions before the cells were cultured in RPMI-1640 with 10% serum replacement (10828028, Gibco) and 1% penicillin/streptomycin (MRC). Recombinant mouse M-CSF (50 ng·mL⁻¹, Life Technologies)/GM-CSF (50 ng·mL⁻¹, Thermo) was used for macrophage polarization. A recombinant mouse FetA protein (10 ng·mL⁻¹, R&D) was added to GM-CSF to test whether FetA can change the polarization of macrophages. F4/80⁺ macrophages were further analyzed by flow cytometry using an anti-CD206 antibody diluted with flow cytometry buffer (0.5% BSA + 0.09% sodium azide in PBS) (see Fig. S21). Flow cytometry data were analyzed with FlowJo software (TreeStar, Inc.) or CytExpert (4.0 version, Beckman).

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).