Bone Marrow Derived Dendritic Cell Isolation and Culture

BMDCs were obtained from 6- to 8-week-old C57BL/6 mice. Mouse tibias and femurs were removed and flushed with cold PBS through a 70-μm-wide cell strainer. The cells were pelleted by centrifugation for 5 min at 450 × g and resuspended in RPMI 1640 medium supplemented with 10% HI FBS, 2 mM l-glutamine, 0.4 mM sodium pyruvate, 50 μM 2-mercapthoethanol, and 20 ng/mL mGM-CSF. Then, the cells were seeded in 100 × 20 mm nontreated cell culture dishes in 10 mL of conditioned medium at a density of 9 × 10⁶ cells per dish, and they were incubated for 8 days at 37 °C in 5% CO₂. Fresh medium was added on days 4 and 7.