Infiniti m200

Nunc polysorb plates (Thermo Scientific, Germany) were coated overnight with indicated amounts of recombinant E ectodomain protein or E-quadruple mutant (in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃ pH 9.6)) per well with gentle agitation at 4°C. The plates were washed three times with 350 μL per well of PBS/Tween (0.05%), followed by blocking with 5% non-fat dry milk powder (200 μL per well) for 2 h at room temperature (RT). After a second wash step, human sera (dilution 1:100 in 5% non-fat dry milk powder, 100 μL per well) were incubated for 1.5 h at RT. The sera were removed by a third wash step and 100 μL of the secondary antibody (1:10.000 diluted HRP-conjugated Goat-anti-Human IgG (Fisher Scientific)) was added for 1 h at RT. After washing, the TMB-substrate (BioLegend, Germany) was added to the wells and the plate was incubated for 30 min at RT in darkness. To stop the reaction, 1 M H₂SO₄ was added, followed by measurement at 450 nm and 520 nm (reference wavelength) in an ELISA Reader (Infiniti M200, Tecan). All antibody tests were performed in duplicates in at least two independent experiments.
Equal loading of wild type and mutant E protein was verified using the humanized E16 monoclonal antibody (dilution 1:1000), which targets an epitope on domain III of the E protein, distant from the fusion loop [23 (link)] (data not shown).

Reactive oxygen species production in N. caninum tachyzoites-stimulated caprine monocytes was determined by 2,7 dichlorofluorescein diacetate (DCFH-DA, Sigma). Briefly, caprine monocytes were incubated with DCFH-DA (10 µM, 15 min) prior to the stimulation with vital N. caninum tachyzoite (ratio: 1:3 or 1:6, 180 min, 37°C). Monocytes stimulated with zymosan (1 mg/ml, Sigma-Aldrich) served as positive controls. Unstimulated monocytes cultured in plain medium alone served as negative controls. In parallel settings, the cells were pretreated with the NADPH oxidase inhibitor (DPI, Sigma-Aldrich) for 30 min before N. caninum stimulation. Finally, the samples were washed three times with phenol red-free RPMI 1640 medium and measured by using a fluorometric plate reader Infiniti M200 (TECAN, Austria) and flow cytometry at 488 nm excitation/525 nm emission wavelength.

Reactive oxygen species (ROS) generation was measured using an oxidation-sensitive fluorescent probe, 2,7-dihydrodichlorofluorescein diacetate (DCFH-DA; Molecular Probes). Cells were incubated with the probe at a final concentration of 10 μM for 30 min. The fluorescence intensities (excitation 492 nm, emission 525 nm) were evaluated in an Infiniti M200 microplate reader (Tecan).