Cd86 pe gl 1

Collagenase/DNAse-treated DLN cell suspensions or mASC-cultured DCs were preincubated with anti-FcγRII/FcγRIII mAb at 2.5 μg/mL (2.4G2; BD Pharmingen) and 7-aminoactinomycin D (2 μg/mL; Sigma Aldrich) and then stained with the following antibodies: CD11c-APC (N481; eBioscience), CD40-PE (2/23), CD80-PE (16-10A1), and/or CD86-PE (GL-1) (BD Pharmingen). For intracellular cytokine staining cells were fixed, permeabilized, and stained for IFN-γ-FITC (XMG1.2), IL-17-PE (TC11-18H10), TNF-α-PE (MP6-XT22), IL-4-PE (BVD4-1D11), and IL-10-PE (JES5-16E3) (all from BD Biosciences, San Diego, CA) using the BD cytofix/cytoperm kit (BD Biosciences) according to the manufacturer's instructions. Each sample was stained with appropriate isotype controls. For the analysis of foxp3 expression cells were stained with CD4-FITC (GK1.5; BD Biosciences) and subsequently processed for foxp3 staining using the foxp3 staining kit (eBioscience) according to the manufacturer's instructions. Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences).

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.