Quadromacs column separation kit

Non-parenchymal cells from mouse liver were isolated by 2-step collagenase perfusion (24 (link)). Macrophage positive selection from non-parenchymal cells was performed using QuadroMACS column separation Kit (Miltenyi Biotech, Cambridge, MA). Anti-mouse F4/80 antibody (Biolegend, San Diego, CA) and specific microbeads were used according to the manufacture’s instruction. After the column separation of macrophages, cytospin was performed. Cells were centrifuged at 500 rpm for 5 minutes on glass slides followed by fixation with 4% paraformaldehyde for 10 minutes. Rat Anti-mouse F4/80 antibody (AbD Serotec, Raleigh, NC) was used for immunofluorescent staining as described elsewhere (25 (link)).
Additional methods in online supplement

Parenchymal and non-parenchymal cells from mouse liver were isolated by a 2-step collagenase perfusion (16 (link)). The parenchymal cell fraction, which consists mostly of hepatocytes, also contains hepatic stellate cells due to their close physical association with hepatocytes in vivo. Positive selection of CD45+ cells and EpCAM+ cells from non-parenchymal cells was performed using the QuadroMACS column separation Kit (Miltenyi Biotech, Cambridge, MA) (10 (link)). Cells were then purified using the Dead Cell Removal Kit (Miltenyi Biotech). Anti-mouse CD45 antibody, anti-mouse EpCAM antibody (Biolegend, San Diego, CA) and microbeads were used according to the manufacturer’s instructions. To assess the purity of EpCAM+ cells, low expression of CD68 (Kupffer cells) and Tie2 (endothelial cells) was confirmed by qRT-PCR.
Additional methods are available in the online Supplementary Methods.