Novostar microplate reader

For analyzing the effects of the FLAP antagonists on cell viability, cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/mL, 20 μL; Sigma-Aldrich, Munich, Germany) for 2–3 h at 37°C (5% CO₂) in the dark. The formazan product was solubilized with sodium dodecyl sulfate (SDS, 10% in 20 mM HCl), and the absorbance was monitored at 570 nm (Multiskan Spectrum microplate reader, Thermo Fisher Scientific, Schwerte, Germany). Staurosporine (1 µM) was used as the positive control. For analyzing the effects of the FLAP antagonists on cell integrity, the release of lactate dehydrogenase (LDH) was assessed using CytoTox 96^® Non-Radioactive Cytotoxicity assay according to the manufacturer´s instructions (Promega, Mannheim, Germany). After treatment, the cells were centrifuged at 400 g (5 min, 4°C), and the supernatants were diluted to suitable LDH concentrations. The absorbance was then measured at 490 nm using a NOVOstar microplate reader (BMG Lab Technologies GmbH, Offenburg, Germany). Cell integrity was calculated according to the manufacturer´s guidelines. Triton X-100 (0.9%, v/v) was used as the positive control.

Adherent M1 and M2 (5 × 10⁶ cells) were pre-stained with Fura-2/AM (2 µM) for 45 min at 37 °C in the dark. After two washing steps, cells were resuspended in PG-BSA buffer (PBS, 0.1% glucose, 0.1% fatty acid-free BSA) at a density of 1.25 × 10⁶/ml. In total, 200 µl of the cell suspension were transferred into a 96-well plate and 1 mM CaCl₂ was added. After 10 min, 1 µM fMLF, 2 µM ionomycin, E. coli (macrophages: E. coli = 1:50) or vehicle (PG-BSA) was added. The signal was monitored in a thermally (37 °C) controlled NOVOstar microplate reader (BMG Labtechnologies GmbH, Offenburg, Germany; emission at 510 nm, excitation at 340 nm (Ca²⁺-bound Fura-2) and 380 nm (free Fura-2)). After cell lysis with Triton X-100, the maximal fluorescence signals were monitored, and after chelating Ca²⁺ with 20 mM EDTA, the minimal fluorescence signals were recorded. The ratio of signals obtained with Triton X-100 subtracted by the signals obtained at basal fluorescence intensity (shown as Δratio) of each experiment was set to 100%.

To determine the effect of ATP (Sigma) and/ or IL‐33 (50 ng/ml) (PeproTech) on the intracellular Ca²⁺ concentration [Ca²⁺]_I, 5x10⁶ BMMCs were resuspended in KREPS‐HEPES buffer (20 mM HEPES pH 7·4,135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0·4 mM KH₂PO₄ and 5·5 mM glucose) and incubated with 0·2 µM Fura‐2/AM (Thermo Fisher Scientific, Waltham, MA) for 30min (37°celsius, 5% CO₂). After centrifugation, cells were recovered in KREBS‐HEPES buffer supplemented with 1 mM CaCl₂ and diluted to a final density of 1 × 10⁶ cells/ml. Cells were transferred onto black 96‐well microplates PS F‐bottom (Greiner bio‐one), and the signal was monitored by a NOVOstar microplate reader (BMG Labtechnologies GmbH) at 37°C : emission at 510 nm, excitation at 340 nm (Ca²⁺‐bound Fura‐2) and 380 nm (free Fura‐2). By subsequent cell lysis with triton X‐100, the maximal fluorescence signals were determined, followed by chelating Ca²⁺ with 20 mM EDTA to assess the minimal fluorescence.