Dendritic Cell Generation From Mouse Bone Marrow

Mouse bone marrow (BM) -derived DCs were generated as previously reported^{48 (link)}. Briefly, BM progenitors isolated from the femurs and tibias were incubated at 5 × 10⁵ cells/ml in generating medium (RPMI 1640 containing 10% FBS [Hyclone], 2 mmol/L glutamine, 25 mmol/L HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μmol/L 2-mercaptoethanol and 20 ng/ml of mouse GM-CSF (PeproTech) at 37 °C in a humidified incubator with 5% CO₂ for 6 d to generate immature DCs. Subsequently, immature DCs were incubated in fresh culture medium at 5 × 10⁵ cells/ml in the absence (sham) or presence of HMGN1, R848 (InvivoGen), or LPS (Sigma-Aldrich, as positive control) alone or in combination at specified concentrations for 6–48 h at 37 °C in a humidified incubator with 5% CO₂ before analyzing their function and phenotype. Recombinant HMGN1 used in the present study was generated in insect cells using a baculovirus expressing system and purified as previously described^{27 (link)}.
For the generation of human DCs, peripheral blood monocytes were isolated and cultured in the presence of 50 ng/ml of human GM-CSF (PeproTech) and IL-4 (PeproTech) for 5 to 7 days as previously described^{48 (link)}.