Anti mouse cd11c clone n418

BMDCs were harvested and stained using anti-mouse CD11c (clone N418; Biolegend), anti-mouse CD11b (clone M1/70; Biolegend), MHC class II I-Ab (clone AF6-120.1; Biolegend), CD86 (clone GL-1; Biolegend), CD40 (clone 3/23; Biolegend), and Zombie Fixable Viability dye (Biolegend) in PBS containing anti-mouse CD16/CD32 (clone 93; Biolegend). Cells were washed with FACS buffer (5% FBS, 1 mM EDTA, 0.01% NaN₃ in PBS) and fixed using 1% paraformaldehyde (PFA, Electron Microscopy Services) in FACS buffer.

Tumor lysates were prepared by mincing the tumor in DMEM (Sigma) and incubating in collagenase (Roche) at 37°C for 1 h. After washing in PBS, the cells were filtered through 70‐μm filters (BD Biosciences). 5 × 10 cells were re‐suspended in HBSS (Hank's balanced salt solution, Lonza) supplemented with 0.5% BSA (Sigma). Staining was performed at 4°C for 20 min, with the following antibodies: anti‐mouse CD45 (clone 30‐F11, Biolegend San Diego, CA, USA), anti‐mouse F4/80 (clone BM8, Biolegend San Diego, CA, USA), anti‐mouse cd11c (clone N418, Biolegend San Diego, CA), anti‐mouse I‐A/I‐E (clone M5/114.15.2, Biolegend San Diego, CA, USA); anti‐mouse TNFα (clone MP6‐XT22, BD Pharmingen), anti‐mouse Ly6C (clone HK1.4, eBioscience; San Diego, CA, USA); anti‐mouse NOS2 (clone 6/iNOS/NOS Type II, BD biosciences, San Diego, CA, USA) and mouse IgG2a K (BD biosciences, San Diego, CA, USA). For intracellular staining, Cytofix/Cytoperm and Permwash staining kit (BD Pharmingen) were used. Cells were detected using the BD FACS Canto II cytofluorimeter and analyzed with FlowJo software.

Non-parenchymal liver cells were incubated with monoclonal antibody 2·4G2 for FcR blocking (BioLegend, San Diego, CA, USA) and then exposed at 4°C to a mixture of the following antibodies (dilutions are indicated):anti-mouse CD45 (clone 30-F11, 1:100), anti-mouse/human CD11b (clone M1/70, 1:300), anti-mouse Ly6C (clone HK1.4, 1:300), anti-mouse MHCII (clone M5/114.15.2, 1:200), anti-mouse CD11c (clone N418, 1:100), anti-mouse CD3ϵ (clone 145-2c11, 1:100), anti-mouse CD8a (clone 53-6.7, 1:100), anti-mouse CD4 (clone GK1.5, 1:100), anti-mouse TCRβ (clone 457-597, 1:100) all were purchased from BioLegend, San Diego, CA. Anti-mouse F4/80 (clone REA 126, 1:100) and anti-mouse Tim4 (clone REA999, 1:100) were purchased from Miltenyi Biotech.
For IL-17A staining, cells were first stained for surface markers, then the cells were fixed and permeabilized prior to intra-cellular staining with IL-17 (Clone TC11-18H10.1, 1:50, BioLegend). Cells were analyzed with BD FACS Canto™ II (BD Bioscience) or sorted with a FACSAria flow cytometer (BD Bioscience). Flow cytometry analysis was performed using FlowJo software (TreeStar, Ashland, OR).