Generation of Human Monocyte- and Murine Bone Marrow-Derived Dendritic Cells

Human monocytes (E59; ∼40 × 10⁶ cells) from healthy volunteers (Ethical approval EK 1880/2012 in accordance with the Declaration of Helsinki, Medical University of Vienna, Vienna, Austria; written informed consent was obtained from all volunteers of this study) were thawed at 37°C and placed into AIM V medium (Gibco, Life Technologies, Paisley, United Kingdom) supplemented with 2% human plasma (Octaplas, OP; Octapharma, Zurich, Switzerland). Monocytes were counted and adjusted to a concentration of 0.5 × 10⁶ cells/ml. Dead cells were removed after incubation at 37°C for 1.5 h, while living cells were differentiated with 1000 U/ml GM-CSF and 400 U/ml IL-4. Medium including cytokines was replaced on day 3 and iDCs were harvested on day 5 (Lanzinger et al., 2012).

Murine BMDCs were isolated from C57BL/6 mice (Gallucci et al., 1999). Briefly, femurs and tibias were rinsed with DMEM, supplemented with 10% heat-inactivated fetal calf serum (FCS; PAA, Pasching Austria), 1% penicillin/streptomycin (Gibco), 1% non-essential amino acids (NEAA) (Gibco) and 0.0002% β-mercaptoethanol (Sigma-Aldrich). Erythrocytes were lysed in erolysis buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA; pH 7.2). The bone marrow was washed, and cells were re-suspended in supplemented DMEM. Cells were cultured in 24-well plates (Iwaki, Japan) at a concentration of 1.0 × 10⁶ cells per well in DMEM supplemented with 10% FCS and in the presence of 5 ng/ml recombinant murine IL-4 (eBioscience, San Diego, CA, United States) and 3 ng/ml recombinant murine GM-CSF (BD Pharmingen, San Diego, CA, United States). Half of the medium (including all supplements) was replaced every second day. Immature DCs (iDs) were harvested on day 6, counted and adjusted to a concentration of 1.0 × 10⁶ cells/ml. All mouse experiments were performed in accordance with the institutional guidelines and approved by the Animal Care and Use Committee of the Medical University of Vienna, Austria (GZ: 856861/2013/16).