Amplex red

NADPH oxidase activity was assessed as the release of hydrogen peroxide, determined by the Amplex Red method (Molecular Probes, Life Technologies, Carlsbad, CA, USA) by neutrophils (1x10⁶/mL) stimulated with: fMLF (1 μM), TNFα (10 ng/mL), LPS (20 ng/mL) + LPS-binding protein (LBP) (50 ng/mL, R&D Systems, Minneapolis, MN, USA) or PMA (100 ng/mL, Sigma) in the presence of Amplex Red (0.5 μM) and horseradish peroxidase (1 U/mL). Fluorescence was measured at 30-s intervals for 4 h with the HTS7000+ plate reader (Tecan, Zurich, Switzerland). Maximal slope of hydrogen peroxide release was assessed over a 2-min interval.

We performed hydrogen peroxide emission analysis as previously published (Koh et al., 2019b (link)). Briefly, palmitate or saline treated PPARδ or empty vector (EV) myotubes in 75 t flasks were washed with DPBS and suspended in 0.25% trypsin-EDTA medium. Myotubes were then centrifuged, re-suspended in respiration medium [105 mM K-MES, 30 mM KCL, 10 mM KH2PO4, 5 mM MgCl2-6H2O, 5 mg/ml BSA] pH 7.4 supplemented with 1 mM EGTA (pH 7.3)] including 10 mM Amplex Red, horseradish peroxidase 1 U/ul, superoxide dismutase 10 U/ul, and treated with 500 μg/ml digitonin (∼50% TLC, Sigma, St. Louis, United States) to permeabilize membranes. Glutamate (5 mM, 2 mmol/L) and malate (1 mmol/L) were then added to stimulate H₂O₂ production under state 4 respiration conditions. A fluorolog 3 (Horiba Jobin Yvon, Edison, Piscataway, NJ, United States) spectrofluorometer was used to monitor Amplex Red (Invitrogen, Carlsbad, United States) oxidation in myotubes.

A fluorimetric assay based on Amplex Red and horseradish peroxidase was used as described previously [15 (link), 27 (link)]. One hundred microliter reactions containing 50 mM sodium acetate pH 6.0, 50 μM Amplex Red (Sigma-Aldrich, Saint-Quentin Fallavier, France), 7.1 U ml⁻¹ horseradish peroxidase, 1–10 μM purified GcLPMOs, and 50 μM ascorbate as reductant in water were incubated for 30 min at 30 °C in 96-Well Black Solid Plates (Greiner Bio One, Kremsmünster, Austria). Fluorescence was measured using excitation and emission wavelengths of 560 and 595 nm, respectively, using a Tecan Infinite M200 plate reader (Tecan, Männedorf, Switzerland). The specific activity was calculated from an H₂O₂ calibration curve, and the slope (13,227 counts μmol⁻¹) was used to convert the fluorimeters readout (counts min⁻¹) into enzyme activity. Various polysaccharides (PASC, CMC, avicel, xylan, xyloglucan, chitin, lichenan, curdlan, starch, β-1,3-glucan, glucomannan, and cello-pentaose) were tested to a final concentration of 0.1% (w/v) or 0.4 mg ml⁻¹. For dose–response inhibition assays on PASC and xyloglucan, substrates were added to final concentrations of 0.1, 0.05, and 0.01% (w/v). All measurements were performed in triplicates.

A fluorimetric assay based on Amplex Red and horseradish peroxidase was used as described previously [30 (link), 31 (link)]. The reaction (total volume 100 μl, 30 °C, 30 min) was measured in 100 mM sodium acetate buffer pH 6.0 containing 50 μM Amplex Red (Sigma-Aldrich, Saint-Quentin Fallavier, France), 7.1 U.ml⁻¹ horseradish peroxidase, 0.2 to 4 μM PaLPMO9, and 50 μM ascorbate as reductant in water and fluorescence and was detected using an excitation wavelength of 560 nm and an emission wavelength of 595 nm using a Tecan Infinite M200 plate reader (Tecan, Männedorf, Switzerland). The specific activity was counted from H₂O₂ calibration curve, and the slope (13,227 counts μmol⁻¹) was used to convert the fluorimeters’ readout (counts min⁻¹) into enzyme activity. For inhibition studies, the range of polysaccharides (cello-oligosaccharides DP4-DP6, PASC, CMC, β(1,3;1,4)-glucan from barley, β(1,3)-glucan, lichenan, starch, glucomannan, laminarin, pectin, xylan, and XG) and their cello-oligosaccharides derivatives were added to a final concentration of 0.1 % (w/v) and 3 mM, respectively. All measurements were performed in triplicates.

HRP and Amplex^® Red were purchased from Sigma. The concentration of HRP was determined spectrophotometrically at 403 nm (ε = 1.02 × 105 M⁻¹ cm⁻¹) [42 (link),48 (link)]. Relationship between the yield of oxidation of Amplex^® Red by H₂O₂ in the presence of the HRP, Cu(II)MP-11 and Fe(III)MP-11, was determined by measuring absorbance intensity due of resorufin formed. Under all conditions, 5 mM pH 7.4 HEPES, 20 mM CTAB and 2 mM DODAB vesicles, containing 40 µM Amplex Red, 25 nM HRP or 100 nM Fe(III)MP-11, 20 µL of hydrogen peroxide solution (2–20 µM) was added with a multichannel micropipette. Absorbances at 571 nm were measured after an automatic brief shaking at 30 °C in a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA). The reactions were done in a total volume of 200 μL per microplate well, in triplicate for each H₂O₂ concentration. The concentration of resorufin formed was calculated assuming the molar absorption coefficient at 571 nm of 6.3 × 10⁴ M⁻¹ cm⁻¹ [43 (link),49 (link)].