Generation and Characterization of Bone Marrow-Derived Dendritic Cells

Bone marrow-derived dendritic cells (BMDCs) were generated as previously described (46 (link), 47 (link)). Briefly, cells were isolated from leg bones of euthanized mice and cultured at a density of 1 × 10⁶ cells/mL in differentiation media [Iscove modified Dulbecco medium (Gibco/Life Technologies, CA) containing 10% heat-inactivated FCS, penicillin-streptomycin (100 U/mL), L-glutamine (2 mM), 2-mercaptoethanol (50 µM), recombinant mouse granulocytes macrophage colony-stimulating factor (20 ng/mL), and recombinant mouse IL-4 (10 ng/mL, PeproTech]. Cultures were supplemented with an equal volume of medium (day 3 and day 5), and nonadherent cells were collected on day 6 and plated in six-well tissue culture plates at a density of 2 ×10⁶ cells/well in differentiation media. Nonadherent cells were collected on day 7 for analysis. BMDCs (2−5 × 10⁵) were cultured on 96-well plates and pulsed with HDM (100 µg/mL) in the presence or absence of rSPLUNC1 (5 µg/mL). BMDCs were further processed for RNA isolation and Western blot analysis. In separate experiments, BMDCs were used for XFp assays and extracellular acidification rates (ECARs) measurements.