Generation of Bone Marrow-Derived Dendritic Cells

BMDC were generated as described (28 (link)). Bone marrow from femurs of 6–10 week old mice was flushed out using a 27 G Precision Glide needle (BD Biosciences, Cat. No. 305109). Cells were plated at 1 ×10⁶ cells in 10 ml of media per Petri Dish (Fisherbrand, Cat. No. FB0875711), supplemented with 10 ng/ml of recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) (STEMCELL Technologies, Cat. No. 78206.1) and 2 ng/ml of recombinant mouse IL-4 (Invitrogen, Cat. No. 14-8041-62). An equivalent amount of fresh media containing cytokines was added 3 days after plating, and 50% of media was changed on day 5 after plating. BMDCs were harvested for use on day 6.