Bone Marrow Dendritic Cell Generation

Bone marrow-derived DC (DC) were generated as previously described (45 (link)), with minor changes. In brief, bone marrow was flushed from femurs and tibia from either young BALB/c ByJ or from Il6 ^−/− BALB/c ByJ mice and plated at 8 × 10⁵ cells/ml in complete RPMI 1640 plus 10 ng/ml recombinant murine GM-CSF (PeproTech, Rocky Hill, NJ). On days 3 and 5, half of the culture media was replaced with fresh complete RPMI 1640 + GM-CSF. On day 6 the TLR9 agonist CpG 1826 (1μM; type B unmethylated CpG-oligodeoxynucleotides [ODNs]) (Integrated DNA Technologies, Coralville, IA), was added to the “activated” DC group followed 3 hours later by Poly I:C (10 μg/ml; Invivogen, San Diego, CA), a combination shown to have a synergistic adjuvant effect (31 (link)). On day 7 non-adherent unactivated DC and TLR agonist activated *DC were harvested and enriched by positive selection using directly conjugated CD11c magnetic beads and a MACS column, as per manufacturer’s instructions (Miltenyi Biotec).