Isolation and Culture of Murine Bone Marrow-Derived Dendritic Cells

BMDCs were isolated and cultured as described previously (15 (link), 25 (link), 26) (link). BM was flushed from the tibiae and femurs of C57BL/6 mice and depleted of red blood cells with ammonium chloride. The cells were plated in 6-well culture plates (10⁶ cells/ml; 3ml/well) in OptiMEM (Invitrogen) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μm 2-mercaptoethanol, 10 mM HEPES (pH 7.4), 20 ng/ml rm GM-CSF, and 10 ng/ml rm IL-4 at 37℃ in a 5% CO₂ atmosphere. On culture day 3, floating cells were gently removed, and fresh medium was added. Nonadherent cells and loosely adherent proliferating DC aggregates were harvested for analysis or stimulation on culture day 6. On day 6, ≥ 80% of the nonadherent cells expressed CD11c. The DCs were labeled with a bead-conjugated anti-CD11c mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) to obtain highly purified CD11c-expressing populations for subsequent analyses, which were subjected to positive selection on paramagnetic columns (LS columns; Miltenyi Biotec), according to the manufacturer’s instructions. The purity of the selected cell fraction was ＞ 90%.