Anti mouse il 17a pe

Anti-mouse IL-17A-PE, CD4-APC and IFN-γ-PE-cy7, anti-mouse IgG isotype, anti-human IFN-γ-PE-cy7, CD4-APC and IL-17A-PE and anti-human IgG isotype were obtained from eBioscience. For intracellular expression of IFN-γ and IL-17, CD4+ T cells were incubated for 1 hours with ionomycin (1 μg/mL), PMA (50 ng/mL) and for another 4 hours with brefeldin A (10 μg/ml, Sigma-Aldrich), harvested, washed and fixed before permeabilization.
BMDCs or MD-DCs were stained for 30 minutes at 4 °C with anti-mouse CD86-FITC (eBioscience), CD11c-APC (eBioscience), CD80-PE (eBioscience), CD40-FITC (BioLegend, San Diego, CA, USA), MHCII-PE (eBioscience), anti-human CD86-PE-cy7 (BioLegend), CD40-FITC (BioLegend), HLA-DR-PE-cy5.5 (BioLegend), and CD80-PE (BioLegend).

Naïve CD4⁺ T cells were isolated from the spleen of WT or GPx1^−/− × Cat^−/− mice using a naive CD4⁺ T cell isolation kit (R&D Systems, Minneapolis, MN). For the induction of Th₁₇ cell differentiation, 1×10⁵ naïve CD4⁺ cells were stimulated with soluble anti-CD3e (1 µg/mL) and soluble anti-CD28 (1 µg/mL, e-Biosciences) in the presence of 2×10⁴ CD11c⁺ DCs for 24 hours, and were cultured further for 2.5 days in the presence of TGF-β1 (5 ng/mL) and IL-6 (20 ng/mL) purchased from R&D Systems. For intracellular cytokine staining, cells were re-stimulated for 4 hr with PMA (25 ng/mL) and ionomycin (250 ng/mL, Sigma) in the presence of a protein trapsport inhibitor containing monensin (BD Biosciences). Then, the cells were harvested and stained for intracellular IL-17A and IFN-γ using a commercial kit for fixation and permeabilization ((BD Biosciences), anti-mouse IL-17A-PE (eBioscience) and anti-mouse IFN-γ-FITC (eBioscience). For the induction of iTreg differentiation, 1×10⁵ naïve CD4⁺ cells were stimulated with plate-coated anti-CD3e (50 ng/well) and soluble anti-CD28 (1 µg/mL) in the presence of TGF-β1 (5 ng/ml) and human recombinant IL-2 (10 U/mL, BD Biosciences). After three days of culture, the cells were harvested for intranuclear staining for FoxP3, using a mouse regulatory T cell staining kit.

Rat MOG35–55 peptides were purchased from Biosynth International (Naperville, IL, USA) and the purity of the peptide was >95%. The sequence of MOG35–55 was MEVGWYRSPFSRVVHLYRNGK. FITC anti-mouse CD3, PE/Cy7 anti-mouse CD4, APC/Cy7 anti-mouse CD44, PerCP/Cy5.5 anti-mouse CD11b antibodies, and LEAF Purified anti-mouse IL-6 were purchased from BioLegend (San Diego, CA,USA). Alexa Fluor700 anti-mouse CD45.2, PE anti-mouse CD69, APC anti-mouse CD25, PE anti-mouse IL-17A, and APC anti-mouse IFN-γ antibodies were purchased from eBioscience (San Diego, CA, USA). PE/Cy5 anti-mouse CD8α antibodies were purchased from BD Pharmingen (Basel, Switzerland). Goat anti-mouse ionized calcium-binding adaptor molecule-1 (IBA1) antibody was obtained from abcam (Cambridge, UK). Rat anti-mouse CD45, Rabbit anti-mouse CCL-2 antibody, and CCL-2 (rat recombinant) were purchased from AbD Serotec (Raleigh, NC, USA). Rabbit anti-mouse CCR2 antibody was purchased from Abcam (Cambridge, UK). 2-((1-benzyl-indazol-3-yl) methoxy)-2-methyl propionic acid (Bindarit) was synthesized by and obtained from Angelini (Angelini Research Center-ACRAF, Italy).