Cd11c fitc n418

Inflammatory leukocytes infiltrating into the CNS were isolated using an established protocol (3,4). In short, CNS tissue was minced and leukocytes were isolated using a two‐step Percoll gradient (90% and 63%). The isolated cells were collected and then washed prior to staining. Cells were incubated in an anti‐CD16/32 Fc Block (BD Biosciences, San Jose, CA) at a dilution of 1:200. Cells were stained with fluorescently tagged rat anti‐mouse IgG for the following cell surface antibodies, Ly6G FITC (1A8), CD11b PE (M1/70), CD45 BV510 (30‐F11), CD8 PE‐Cy7 (53–6.7), (BD Biosciences), Ly6C APC (HK1.4), CD4 BV785 (RM4‐5), F4/80 FITC (BM8), I‐A/I‐E APC (M5/114.15.2), CD11c FITC (N418) (Biolegend, San Diego, CA), and CD4 FITC (RM4‐5) or Armenian hamster anti‐mouse IgG for CD80 PerCP‐Cy5.5 (16‐10A1) (BD Biosciences). A full description of gating strategies for flow cytometric staining and intracellular cytokine staining is provided in the Supporting Information Figs. S1–S4.

Bone marrow was isolated from C57BL/6 mice, and the isolated cells were differentiated into DCs according to a previously described protocol, with slight modifications [18 (link)]. These cells were seeded in a 24-well plate in RPMI 1640 (Welgene) with a 5% granulocyte-macrophage colony-stimulating factor (GM-CSF) medium. The medium was replaced every 2 days, and bone marrow-derived dendritic cells (BMDCs) were harvested at day 6.
BMDCs (2 × 10⁵ cells/well) were seeded in a 96-well plate and stimulated with 1, 10, or 100 ng/mL of rIL-33 at 12 h prior to infection with the PR8 virus (1, 10, or 50 multiplicity of infections [MOIs]). At 24 h post-stimulation or 48 h post-infection, the cells and the culture medium were harvested. The production of IL-12p40 in the culture medium was measured by ELISA (BD Bioscience), according to the manufacturer’s protocol. The expression of surface markers, such as CD11c-FITC (N418, Biolegend), CD86-PE (GL1, BD Bioscience), and MHCII-PerCP-cy5.5 (M5/114.15.2, BD Bioscience), in harvested BMDCs were analyzed by flow cytometry.

Spleens were minced through a 70 μm sieve and leukocytes were obtained after lysing red blood cells with ACK buffer. Livers were minced through a 100 μm cell strainer and digested for 30 min at 37 °C with rotation in HBSS (Ca²⁺, Mg²⁺) buffer containing 1 mg/ml (335 U/mg) collagenase IV (Sangon Biotech, Shanghai, China) and 200 μg/ml (100 Kunitz U/ml) DNase I (Sangon Biotech, Shanghai, China). Liver leukocytes were separated by percoll gradient centrifugation (GE Healthcare, Freiburg, Germany). The cells were then resuspended in RPMI-1640 at 1 × 10⁶/ml, and surface staining was performed as previously described [16 (link)].
F4/80 and CD11b were used to stain macrophages, CD11c and CD11b for DCs, and Ly6G and CD11b for granulocytes. CD11c-FITC (N418; Biolegend), Gr1-PE (RB6-8C5; Biolegend), CD11b-PerCP-Cy5.5 (M1/70; eBioscience), F4/80-APC (BM8; Biolegend), and CD3-Pacific Blue (145-2C11; eBioscience) antibodies were utilized. As for intracellular iNOS-PE (W16030C; Biolegend) staining, the fixation and permeabilization steps were followed by Intracellular Fixation & Permeabilization Kit (eBioscience). Flow cytometric acquisition was performed with a BD FACSVerse flow cytometer, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).