Evaluating Inflammatory Biomarkers and Oxidative Stress

The concentrations of TNF-α, IL-6, IL-10, IL-17a and interferon-gamma (IFN-γ) (all from eBioscience, USA) in the supernatants of serum-stimulated cells and serum from participants were quantified by ELISA kits, according to the manufacturer’s protocols. Lipid peroxidation was measured through the TBARS in PBMCs following a previously described protocol^{69 (link)} and expressed in MDA nmol/mL. Total thiol concentrations were measured in PBMCs using 5–5′-dithio-bis(2-nitrobenzoic acid) reagent⁹ . ROS production was evaluation in the cell lysate through the fluorescence intensity of the redox-sensitive dye 2′,7′-dichlorodihydrofluorescein diacetate (DCFH, 100 µM, Sigma-Aldrich) (excitation and emission wavelengths of 480 and 535 nm, respectively) using SpectraMax M2e (Molecular Devices, USA).