Generation of Bone Marrow-Derived Dendritic Cells

BMDCs were prepared as previously described.^{15 (link),35 (link)} In brief, single-cell suspensions of bone marrow cells (1 × 10⁶ cells per ml) derived from C57BL/6 mice were cultured in RPMI-1640 medium containing 10% FBS, 1 mM sodium pyruvate, 20 mM HEPES, 2 mM l-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol supplemented with mouse granulocyte-macrophage colony-stimulating factor (GM-CSF; 20 ng/ml; PeproTech). On days 3 and 5, half of the medium was replaced with fresh medium. Nonadherent BMDCs were harvested for a stimulation assay on day 7 in the presence of fresh GM-CSF. The percentage of CD11c⁺ cells in the BMDCs was evaluated by flow cytometry and found to be greater than 70%.