Mber: SYXK-2010-0098). The murine melanoma cell line B16F10 (ATCC, CRL-6475) expressing OVA (B16-OVA) was cultured in 10 FCS RPMI (Sigma Aldrich, 2 mM glutamine, 1 M HEPES, 100 mg/ml streptomycin and 100 U/ml penicillin, two mM 2-mercaptoethanol). All cell lines had been cultured at 37uC in a humidified atmosphere of 5 CO2 and air.Ex vivo T cell stimulation and intracellular cytokine stainingSingles cells prepared from spleens have been stimulated in vitro for 4 hrs with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 mM; each from Calbiochem), with the addition of monensin option (Biolegend) through the final 2 hrs. Cells had been then stained for surface markers. For intracellular cytokine staining, cells were stained for surface molecules initially, then fixed and permeabilized with Cytofix/Cytoperm buffer (eBioscience) and subsequently incubated with anti-cytokine antibodies in Perm/Wash buffer (eBioscience) for 30 min. Handle staining with isotype handle IgGs was performed in all experiments.ELISAIL-6, IL-12p70, IL-23 (p19/p40) and TNF-a concentrations inside the sera have been measured in triplicate employing normal ELISA kits (Biolegend).Chemical substances and cytokinesFucoidan of Fucus vesiculosus and chicken ovalbumin (OVA) were obtained from Sigma-Aldrich. The endotoxin levels in commercially obtained fucoidan have been evaluated making use of a Limulus amebocyte lysate (LAL) assay kit (Lonza). Fucoidan and OVA applied in all experiments contained significantly less than 0.1 endotoxin unit/ml. The H2Kb restricted peptide OVA25764 (SIINFEKL) was bought from Chinapeptides (China).CA XII Inhibitor Accession Real-time PCRTotal RNA was reverse-transcribed into cDNA working with Oligo (dT) and M-MLV reverse transcriptase (Promega). The cDNA was subjected to real-time PCR amplification (Qiagen) for 40 cycles with annealing and extension temperature at 60uC, on a LightCycler 480 Real-Time PCR System (Roche). Primer sequences are: mouse bActin forward, 59-TGGATGACGATATCGCTGCG-39; reverse, 59-AGGGTCAGGATACCTCTCTT-39, IL-6 forward, 59-AACGATGATGCACTTGCAGA-39; reverse, 59-GAGCATTGGAAATTGGGGTA-39, IL-12p40 forward, 59-CACATCTGCTGCTCCACAAG-39; reverse, 59- CCGTCCGGAGTAATTTGGTG39, IL-23p19 forward, 59-CTC TCG GAATCTCTGCAT GC-39; reverse, 59-ACCATCTTCACACTGGATACG-39, TNF-a forward, 59-CCTTTCACTCACTGGCCCAA-39; reverse, 59-AGTGCCTCTTCTGCCAGTTC-39 T-bet forward, 59-CAACAACCCCTTTGCCAAAG-39; reverse, 59-TCCCCCAAGCATTGACAGT-39, GATA3 forward, 59-CGGGTTCGGATGTAAGTCGAGG-39; reverse, 59- GATGTCCCTGCTCTCCTTGCTG-39, RORct forward, 59-CCGCTGAGAGGGCTTCAC-39; reverse 59TGCAGGAGTAGGCCACATTACA-39, IFN-c forward, 59-GGATGCATTCATGAGTATTGC-39; reverse, 59-CTTTTCCGCTTCCTGAGG-39, IL-4 forward, 59-ACAGGAGAAGGGACGCCAT-39; reverse 59-GAAGCCCTACAGACGAGCTCA-39, IL17A forward, 59-GCGCAAAAGTGAGCTCCAGA-39; reverse 59ACAGAGGGATATCTATCAGGG-39.AntibodiesIsotype handle antibodies (Abs) (IgG1, IgG2a or IgG2b), CD11c (HL3), CD4 (GK1.5), CD8a (YTS169.4), CD40 (3/23), CD80 (16-10A1), CD86 (GL-1), anti-IL-4 (11B11) and anti-IL-12/ 23p40 (C17.eight) have been from BioLegend; anti-MHC class I (AF688.5.three), anti-MHC class II (M5/114.15.two), anti-IFN-c (XMG1.2), anti-IL-17 (TCC11-18H10.1) and anti-TNF-a (MP6-XT22) were from eBioscience.Flow cytometry analysisCells have been washed with phosphate buffered saline (PBS) containing 0.five BSA, pre-incubated for 15 min with unlabeled isotype Caspase Activator site manage Abs, and after that labeled with fluorescence-conjugated Abs by incubation on ice for 30 min followed by washing with PBS. Cells have been analyzed on a FACS Aria II (Becton Dickinson) and FlowJo 8.six s.