L 012

Dimethyl sulfoxide (DMSO), N-formyl-Met-Leu-Phe (fMLF), phorbol 12-myristate 13-acetate (PMA), Histopaque 1077, α-terpineol, myrtenol, and γ-terpinene were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Geraniol, germacrene D, α-humulene, and β-caryophyllene were from Cayman Chemicals (Ann Arbor, MI, USA), and (-)-terpinen-4-ol was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan). n-Hexane was purchased from Merck (Darmstadt, Germany). Fluo-4AM was purchased from Invitrogen (Carlsbad, CA, USA). L-012 was purchased from Tocris Bioscience (San Diego, CA, USA). Hanks’ balanced salt solution (HBSS; 0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 5.56 mM glucose, and 10 mM HEPES, pH 7.4) was purchased from Life Technologies (Grand Island, NY, USA). HBSS without Ca²⁺ and Mg²⁺ is designated as HBSS⁻; HBSS containing 1.3 mM CaCl₂ and 1.0 mM MgSO₄ is designated as HBSS⁺.

For measurement of ROS production, 30,000 BMDCs were seeded per well of a white 96 well plate (Greiner) in a volume of 50 µl in RPMI medium without FCS and phenol red (Gibco). Cells were allowed to settle for 1 h. To induce ROS production, cells were treated with 50 µg/ml depleted Zymosan, 50 µg/ml Zymosan, 200 µg/ml Curdlan, MOI 10 HKCA or 500 ng/ml LPS, which were added to the cell suspension in a total volume of 50 µl as a twofold concentrated solution. Additionally, these solutions were supplemented with 100 µM L-012 (Tocris) serving as luminescent probe for ROS detection. In blocking experiments, cells were incubated with 10 µg/ml anti-Dectin-1 (2A11, Biorad) for 30 min prior to ligand application. Luminescent signals were detected either employing Biotek Synergy, Infinite M Plex (Tecan), Victor^{3 (link)} 1420 (Perkin Elmer) or CLARIOstar Plus (BMG labtech) multi-mode readers every 2.5 min for a total of 90 min. ROS production was quantified as Area under the curve (AUC), which was calculated using GraphPad Prism software.

HC-030031, supplied by Hydra Biosciences (Cambridge, MA, USA), was suspended in 0.5% methylcellulose and administered i.p. in a volume of 10 ml/kg. L-012 was purchased from Tocris Bioscience (Avonmouth, Bristol, UK) and dissolved in ultra pure H₂O for subcutaneous injection. Catalase from bovine liver (2,000–5,000 IU/mg protein) supplied as a range of activity, was purchased from Sigma-Aldrich (St Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA) for local injection at 1 mg/ml and pH 7.0. For local injections of HC-030031, drug was dissolved in 10% dimethylsulfoxide (DMSO; Molecular probes, Eugene, OR, USA) in PBS. H₂O₂ was purchased from Sigma-Aldrich (St. Louis, MO, USA), and diluted in SIF or PBS.