Amplex red hydrogen peroxide peroxidase assay

H₂O₂ accumulation in leaves was measured by using a commercial kit (Amplex Red hydrogen peroxide-peroxidase assay, Molecular Probes/Invitrogen, Eugene, OR, USA) with few modifications. Briefly, 500 μL of 50 mM sodium phosphate buffer at pH 7.4, containing 50 μM of Amplex Red reagent and 0.05 U mL^-1 of horseradish peroxidase, was added to approximately 40 mg of frozen leaf tissue and incubated for 30 min at room temperature in darkness. Then, samples were centrifuged at 12000 g for 12 min at 4°C and 50 μL of supernatants were transferred into new opaque tubes. Absorbance at 560 nm was measured by using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The concentration of H₂O₂ in each sample was determined from a standard curve consisting of 0, 0.5, 1, 3, 6, and 9 μM H₂O₂. After absorbance measurements, H₂O₂ accumulation per mg of fresh weight was calculated.

BMDM and PM were seeded at a density of 250,000 cells/well in a 96-well plate with a µClear® bottom (Greiner Bio-one, 655090). The hydrogen peroxide (H₂O₂) released was quantified using Amplex® Red Hydrogen Peroxide/Peroxidase Assay (Thermo Fisher Scientific, A22188). Experiments were carried out in 100 µL of PBS pH 7.4 containing 11.1 mM glucose, 25 μM Amplex® Red Reagent (Thermo Fisher Scientific, A12222), and 0.2 U/ml Pierce^TM Horseradish Peroxidase (HRP, Thermo Fisher Scientific, 31490) in the dark at 37°C for 40 min. The resorufin product formed was monitored by fluorescence spectroscopy (excitation energy at 530 nm and emission energy at 590 nm) every 10 min in Spectramax M3 (Molecular Devices). Background signal was determined by adding 500 U/ml Catalase from Bovine Liver (Sigma-Aldrich, E3289) in one well to convert hydrogen peroxide into water, abolishing the non-specific signal. Cells were incubated with 1 µM FCCP to eliminate the mitochondrial contribution to H₂O₂ generation. Thus, the mitochondrial H₂O₂ released was calculated by the difference between the total H₂O₂ released and FCCP-treated cells. Fluorescence data were converted into H₂O₂ concentration using a standard curve with known H₂O₂ concentrations. H₂O₂ released rates (H₂O₂ ηM/min) were further normalized by the amount of DNA in each sample after staining cells with violet crystal.

For this assay, 200 μL of PBS was treated with CAP for 5, 10, 20, 40, and 80 s. We performed each examination with 3.0, 3.5, 4.0, 4.5, and 5.0 of SLM gas flow. The PBS was treated analogously to the cell suspensions in our other experiments. The treated PBS was diluted at 1:100 and an Amplex Red hydrogen peroxide/peroxidase assay (Thermo Fisher Scientific, Waltham, MA, USA) was carried out according to the manufacturer’s instructions. The data were analyzed using linear regression analysis.

ROS/RNS analysis was performed using an OxiSelect In Vitro ROS/RNS Assay (Cell Biolabs, San Diego, California, USA) as per manufacturer’s instructions. The mean fluorescent intensity at 480 nm (excitation) and 530 nm (emission) were measured using a fluorescence plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA).
A secondary method of hydrogen peroxide analysis was performed using an Amplex Red Hydrogen Peroxide/Peroxidase Assay (ThermoFisher Scientific, Waltham, Massachusetts, USA) as per manufacturer’s instructions. The mean fluorescent intensity at 530 nm (excitation) and 590 (emission) were measured using a fluorescence plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, USA).

H₂O₂ synthesized by cardiomyocytes and released into the supernatant was quantified using Amplex red Hydrogen Peroxide/Peroxidase assay according to manufacturer’s instructions (Thermo Fisher Scientific). Briefly, cardiomyocytes (1.5*10⁴) treated (or not) with Co₃O₄-NPs were resuspended in 20 μl Krebs–Ringer phosphate glucose buffer (KRPG; 145 mM NaCl, 5.7 mM sodium phosphate, 4.86 mM KCl, 0.54 mM CaCl₂, 1.22 mM MgSO₄, 5.5 mM glucose, pH 7.4) and added to 100 μl of reaction mixture (containing 50 μM Amplex® Red reagent and 0.1 U/mL horseradish peroxidase in KRPG), prewarmed at 37 °C for 10 min. Samples were incubated at 37 °C for 1 h and the fluorescence was measured using a fluorescence microplate reader (TECAN SpectraFluor Plus). In the presence of peroxidase, the Amplex Red reagent reacts with H₂O₂ and produces resorufin, a red-fluorescent oxidation product (with excitation/emission at 535/595 nm). As positive control, cardiomyocytes treated for 20 min with 0.003% H₂O₂ (0.88 μM) were also analyzed with Amplex red Hydrogen Peroxide/Peroxidase assay.