Isolation and Analysis of Liver Immune Cells

We isolated primary hepatocytes and NPCs as previously described (20 (link), 49 (link)). Briefly, we anesthetized mice and digested livers by perfusion of EGTA buffer and collagenase buffer (MilliporeSigma, C5138) through the inferior vena cava, purified hepatocytes with Percoll, and concentrated the remaining NPCs by Nycodenz density centrifugation. We analyzed NPCs by multicolor flow cytometry using an LSRFortessa (BD Biosciences). Briefly, we centrifuged isolated cells at 450g for 5 minutes at 4°C, washed in cold staining buffer (PBS, 2% BSA), resuspended 1 × 10⁶ to 10 × 10⁶ NPCs in Zombie Aqua Fixable Viability Dye (BioLegend, 423101) diluted 1:1,000 in PBS, and then incubated for 15–30 minutes at room temperature in the dark. After another wash, we incubated NPCs with TruStain FcX Fc receptor blocker (BioLegend, 101319) for 5 minutes, then with fluorochrome-conjugated antibodies against mouse CD45 (BioLegend, 103157), CD11b (BioLegend, 101239), CD11c (BioLegend, 117329), Ly6C (BioLegend, 128011), Ly6G (BioLegend, 127617), F4/80 (BioLegend, 123130), CD3 (BioLegend, 100236), B220 (BioLegend, 103224), and NK1.1 (BioLegend, 156508) diluted at 1:200 for 20 minutes at 4°C in staining buffer. Gating strategy is shown in Supplemental Figure 1. After staining, we fixed cells with 4% paraformaldehyde for 15 minutes at room temperature, washed, and then resuspended in staining buffer prior to sample acquisition. Total NPCs were further fractionated by FACS, using vitamin A fluorescence of HSCs as previously described (49 (link)), or antibody-based cell sorting of lymphoid cells with CD45-APC (BD Biosciences, 559864), myeloid cells with CD11b-FITC (BD Biosciences, 553310), and cholangiocytes with EpCAM-PE (Invitrogen, 12579182). We analyzed data using FCS Express7 (De Novo Software).