Amplex ultrared hydrogen peroxide

Hydrogen peroxide (H₂O₂) concentrations in aorta homogenates were determined by fluorescence in black microplates (Nunclon^® Surface, Thermo Fisher Scientific^®, Vantaa, Finland), using the Amplex^® UltraRed Hydrogen Peroxide (10-acetyl-3,7-dihydroxiphenoxazine) Assay (Invitrogen^®, Paisley, United Kingdom). This reagent, in the presence of peroxidase (horseredish peroxidase, HRP), stoichiometrically reacts with H₂O₂ to form a red-fluorescent oxidation product, resorufin. A standard curve of H₂O₂ was prepared by dilution into reaction buffer, with concentrations ranging from 0 to 10 μmol.L^-1. Next, 50μL from the prepared curve points or from the aorta homogenate samples (or plasma) were plated in duplicate, with the addition of 50μL of working solution/HRP for beginning the reaction. The microplates were incubated at room temperature for 120 minutes, protected from light, and read at wave lengths of 530 nm and 590 nm for excitability and emission, respectively, in a microplate reader (Tecan 200 Infinite^® 200 PRO plate reader, Männedorf, Switzerland).

A piece of frozen eWAT was homogenized in a RIPA lysis buffer (pH 7.5; Cell Signaling Technology®, Beverly, MA) containing protease and phosphatase inhibitor cocktails (Roche®, Mannheim, Germany). Total protein levels were determined by the Bradford assay [21 (link)]. The eWAT catalase activity was measured according to Xu and colleagues [22 (link)], and enzyme activity was expressed in μmol·min·mL⁻¹ per eWAT protein (mg·mL⁻¹) [21 (link)]. Total superoxide dismutase (SOD) activity was assessed with a commercial colorimetric kit (#19160, Sigma®, Seelze, Germany) following the manufacturer's instructions. Lipid peroxidation in eWAT was determined by measuring the thiobarbituric acid reactive substances (TBARS), as a marker of oxidative stress, mainly malondialdehyde (MDA). The quantification was performed according to Ohkawa et al. [23 (link)] with modifications, as previously described [24 (link)]. Data were normalized per total protein concentration, measured by Bradford [21 (link)] and expressed as nM·mg protein⁻¹.
Hydrogen peroxide (H₂O₂) was measured by fluorescence using the Amplex® UltraRed hydrogen peroxide (10-acetyl-3,7-dihydroxiphenoxazine) assay (Invitrogen®, Paisley, United Kingdom) as described [24 (link)].