Cd14 sa14 2

Single-cell suspensions of liver or spleen cells or cultured HSC were incubated with Fc blocking reagent (Biolegend) for 10 min followed by 30 min incubation with fluorescently-conjugated mAbs directed against mouse CD11b (M1/70), CD11c (N418), CD45 (30-F11), F/480 (BM8), Gr1 (RB6-8C5), CD115 (AFS98), and MHC II (M5/114.15.2; all Biolegend). Cells were also tested for expression of Dectin-1 (2A11; Abcam), TLR4 (SA15-21; Biolegend), CD14 (Sa14-2; Biolegend) and TLR2 (6C2; eBiosciences). Human liver NPC and PBMC were stained with mAbs directed against CD45 (HI30), CD14 (M5E2; both Biolegend), or Dectin-1 (259931; R&D). For intracellular cytokine staining, liver NPC were incubated for 4 hours with Brefeldin A (1:1000) before permeabilization of cells and staining using fluorescent conjugated mAbs against murine TNF-α (MP6-XT22;) or IL-6 (MP5-20F3; both Biolegend). Experiments were performed using the LSRII cytometer (BD Biosciences) and analysis was done using FlowJo software (Tree Star).

BAL cells were purified by centrifugation on Percoll to remove surfactant and debris ^{31 (link)}. BAL cells or BMDMs were immunostained to detect CD115 (AFS98), F4/80 (BM8) (eBioscience), CD3 (145-2C11), CD11b (M1/70), CD11c HL3), CD16/32 (2.4G2), CD19 (1D3), CD64 (X54-5/7.1.1), CD131 (JORO50), Ly6G (1A8), CD45.1 (A20), CD45.2 (104), MHC class II (I-A/I-E) (M5/114.15.2), SiglecF (E50-2440) (BD Biosciences), CD14 (Sa14-2) (BioLegend), CD68 (FA-11) (AbD Serotec), and MerTK (108928) (R&D Systems), as previously described^{1 (link)}, evaluated by flow cytometry using a FACSCaliber or FACSCanto flow cytometers (both from BD Biosciences, San Jose, CA), and the results were analyzed using CellQuest, FACSDiva (BD Biosciences), or FlowJo software (Tree Star). For intracellular staining of CD68, Leucoperm (AbDSerotec) was used as directed by the manufacturer.