Tlr4 sa15 21

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).

Peripheral blood was collected retro-orbitally from isoflurane-anesthetized mice. The blood was treated with ACK lysis buffer for 5 min to remove RBCs, leaving the peripheral blood leukocytes (PBLs). To determine expression of cell surface molecules, we incubated PBLs with mAb at 4°C for 20–30 min, and cells were subsequently fixed for 10 min using Cytofix Solution (BD Biosciences). The following mAb clones were used to stain processed samples: CD11a (M17/4; eBioscience), TLR4 (SA15-21; BioLegend), CD3ε (145-2C11; BioLegend), NK1.1 (PK136; eBioscience), NKp46 (29A1.4; BioLegend), F4/80 (BM8; BioLegend), TLR2 (CB225; BD Bioscience), CD19 (6D5; BioLegend), CD11c (HL3; BD Biosciences), CD4 (GK1.5; BioLegend), Ly6G (1A8; BioLegend), CD127 (A7R34; BioLegend), Ly6C (HK1.4; BioLegend), and CD8α (53-6.7; BioLegend). Flow cytometry data were acquired on a Cytek Aurora (Cytek, Bethesda, MD) and analyzed with FlowJo software (Tree Star, Ashland, OR).