Quantifying Oxidative Stress Responses

Donors were submitted to treatment with and without compounds and incubated at 37°C for 5 h with shaking. After that, the ROS detection assay was done as described by Castro-Alférez et al.,^{27 (link)} with adjustments. Measurements of chemical hydrolysis of the probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; Sigma–Aldrich, St. Louis, USA) in the fluorescent compound 2,7-dichlorofluorescein (DCF) were detected by fluorescence spectroscopy at 522 and 498 nm emission and excitation wavelengths, respectively. Fluorescence detection was performed in 96-well solid-bottom black plates using the TECAN 200 Pro (Tecan, Männedorf, Switzerland) fluorimeter. H₂O₂ at 2.5 mM was used as a positive control for the experiment.