Cytokine Profiling of Mouse Bone Marrow-Derived Dendritic Cells

Mouse bone marrow-derived dendritic cells (BM-DC) derived from naïve BALB/c mice (8 weeks of age; n = 3) were prepared as previously described [34 (link), 39 (link)]. Briefly, bone marrow precursors isolated from femurs and tibias were seeded at 2 x 10⁵ cells/ml in RPMI 1640 culture medium containing 10% FCS, 150 μg/ml gentamycin, and 20 ng/ml mouse GM-CSF (Sigma-Aldrich, Germany) and incubated for 8 days. BM-DC (10⁶ cells/well) were stimulated with formalin-inactivated B. longum strains Bl 7952 and Bl 372 (10⁷ CFU/well), Pam3CSK4 (1 μg/ml), ultrapure LPS (1 μg/ml, InvivoGen, USA) or left untreated for 18 h. The levels of IL-10, IL-12p70, IL-6 and TNF-α were analysed in supernatants of stimulated cells by ELISA using Ready-Set-Go! kits (eBioscience, USA) according to manufacturer’s instructions. For cell surface marker analysis, BM-DC were labelled for 30 min at 4°C with anti-mouse FITC-conjugated CD11c, APC-conjugated MHC II and PE-conjugated CD40, CD80 or CD86 monoclonal antibodies (eBioscience, USA). The data were acquired on a BD FACSAria III flow cytometer (BD Biosciences, USA) and analysed with FlowJo software 7.6.2 (TreeStar, USA).