Recombinant mouse granulocytes macrophage colony stimulating factor

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.

Femur bones were isolated from euthanized WT mice and cultured at a density of 1 × 10⁶ cells ml⁻¹ in Iscove modified Dulbecco medium (Gibco/Life Technologies, CA) containing 10% heat-inactivated FCS, penicillin-streptomycin (100 U ml⁻¹), L-glutamine (2 mM), 2-mercaptoethanol (50 µM), recombinant mouse granulocytes macrophage colony-stimulating factor (20 ng ml⁻¹) and recombinant mouse IL-4 (10 ng ml⁻¹, Peprotech). Equal volumes of medium were supplemented (day 3 and day 5), and non-adherent cells were collected on day 6 and plated in 6-well tissue culture plates (2 × 10⁶ cells/well) in 2 ml of differentiation media. Cells were stimulated with HDM (100 µg ml⁻¹) alone and with c-di-GMP (10 µM) in the presence of 5% FBS containing media without GMCSF and IL-4 for 16 h.