Multiparametric Phenotyping of Liver Immune Cells

Mouse cell surface molecule and intracellular IFN-γ and FoxP3 staining was performed as described (32 (link)). Liver NPC were treated with FcγR-blocking rat anti-mouse CD16/32 mAb (2.4G2) to prevent non-specific Ab binding. For cell surface staining, the NPC were incubated for 30 min with FITC-, phycoerythrin (PE)-, allophycocyanin (APC) -, Pacific Blue-, or PE-cyanin (Cy)7-conjugated mAbs to detect expression of CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11c (HL3), CD45 (30-F11), H2-K^b (AF6-88.5), NK1.1 (PK136) (BD Biosciences, San Diego, CA), CD19 (eBio 1D3, eBioscience, San Diego, CA) and F4/80 (BM8, BioLegend, San Diego, CA). For intracellular cytokine staining, cells were fixed with 4% paraformaldehyde and permeabilized using 0.1% saponin, then stained with anti-mouse IFN-γ Ab (XMG1.2) (BioLegend). For Foxp3 staining, cells were fixed and permeabilized using Foxp3 Fix Permkit (eBioscience) and stained with anti-Foxp3 mAb (FJK-16s) (eBioscience). Appropriate Ig isotype controls were obtained from BD PharMingen (San Diego, CA). Flow cytometry was performed using a LSR Fortessa flow cytometer (BD Biosciences) and data were analyzed using FlowJo software (version 7.6; TreeStar, Inc., Ashland, OR).