C57BL/6 (B6), 5-Fluoracil datasheet B6.SJL, OT-II, OT-II B6.SJL and clec9aegfp/egfp20 mice were bred at Cancer Research UK in specific pathogen-free conditions. For some experiments, B6 mice were obtained from Charles River. All animal experiments were performed in accordance with national and institutional guidelines for animal care. Culture medium was RPMI 1640 supplemented with penicillin, streptomycin, HEPES, 2-mercaptoethanol, non-essential amino acids,
sodium pyruvate, glutamine (all from Invitrogen) and 10% heat-inactivated FBS (Bioclear). Poly I:C and curdlan were obtained from Amersham and Wako, respectively. OVA323–339 peptide was synthesized and purified by HPLC at Cancer Research UK. Sterile-filtered egg white was prepared as previously described 22. The antibodies used for ELISA, specific for mouse IFN-γ (R4-6A2 and XMG1.2 clones) and mouse IL-17 (TC11-18H10 and TC11-8H4.1 clones) were obtained from BD. Antibodies specific for B220 (RA3-6B2), CD62L (MEL-14), CD25 (PC61), CD44 (IM7), CD4 (RM4-5), CD8α (53-6.7), CD11c (HL3), FcγRIII-II (2.4G2), IFN-γ (XMG1.2), Ly-6G and Ly-6C (RB6-8C5), CD3ε (145-2C11) and CD45.2 (104) were obtained from BD. Anti-CD45.1 (A20), anti-Foxp3 https://www.selleckchem.com/products/abt-199.html (FJK-16s), anti-FR4
(12A5) and anti-IL-17 (TC11-18H10.1) mAb were purchased from eBioscience. Cell suspensions were blocked with 2.4G2, anti-FCγR washed, resuspended in FACS buffer (PBS, 2 mM EDTA, 2% FBS, 0.2% NaN3) containing the appropriate cocktail of antibodies and incubated on ice for 20 min. For intracellular cytokines detection, Fix and Perm® kit (Invitrogen) was used according to manufacturer’s instructions. Foxp3 expression was assessed using anti-rat/mouse Foxp3 staining set (eBioscience). Flow cytometry data were acquired on a FACS Calibur or on a LSR II flow cytometer (BD) and were analyzed using FlowJo software (Treestar). Anti-DNGR-1 mAb (7H11, rat IgG1) was generated as previously described 9. The Avena phytochrome-specific MAC49 clone was used as isotype-matched control. Antibodies were activated Leukotriene-A4 hydrolase with sulfo-SMCC (Pierce) and purified by molecular size
exclusion chromatography. OVA323–339 peptides, with an added cysteine and biotin at the C-terminus (Cancer Research UK), were added and the conjugation reaction was allowed to proceed for 1 h. Conjugates were isolated with GammaBind™ plus Sepharose™ (GE Healthcare). Finally, the number of peptides coupled to each mAb was determined with a Fluoreporter® Biotin Quantitation kit (Invitrogen). The molar ratio between peptides and mAb varied from 1 to 2 but was systematically adjusted between the two antibodies. Mice were injected i.v. with 2 μg of OVA323–339-coupled mAb. Four hours later, or at the indicated time points, splenocytes were separated into two fractions using anti-CD11c microbeads (Miltenyi).
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