Glycan-Based Immune Activation in BM-DCs

After 4 days of derivation, the BM-DCs were stimulated for 1, 3 and 6 h with the following (n = 3 wells/treatment): RPMI-1640, 2.5 doses/well (equivalent to at least 2.5 × 10^3.5 EID₅₀) UV-inactivated IBV (Nobilis IB Ma5 Vet vaccine, MSD Animal Health), 100 µg/mL pustulan from Lasallia pustulata (InvivoGen, San Diego, CA, USA), 500 µg/mL Mannan from Saccharomyces cerevisiae (Sigma-Aldrich), 200 µg/mL chitosan (Sigma-Aldrich, cat. no. C3646), 10 µg/mL furfurman from Malassezia furfur (InvivoGen, cat. no. tlrl-ffm), 50 µg/mL MMG, or 50 µg/mL TDB. MMG and TDB were provided by Statens Serum Institut. Post stimulation, the BM-DCs were washed 3 times in ice-cold PBS, incubated in 10 mM EDTA on ice for 10 min and detached by thoroughly pipetting up and down. Subsequently, the cells were analysed by flow cytometry. The level of endotoxin contamination was determined by providers or previous studies to be insignificant in the glycan-based ligand concentrations used [34 (link),37 (link),38 (link)].