Intracellular ROS Quantification Assay

Intracellular ROS levels were determined by using the ROS molecular probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) (Molecular Probe, Eugene, OR, USA) as previously described with minor modification [24 (link),25 (link)]. In this assay, ROS oxidize H₂DCF, producing the fluorescent compound DCF, the fluorescence levels of which are proportional to the amount of intracellular ROS. Cells were treated as indicated in figure legends and then processed for the intracellular ROS assessment. For the ROS assay, cells were incubated for 30 min with Hank’s Balanced Salt Solution (HBSS) containing 5 µM H₂DCF-DA, then washed twice with HBSS, and then fluorescence was measured by using a GENios plus microplate reader (Tecan, Mannedorf, Switzerland. Excitation and emission wavelengths used for fluorescence quantification were 485 and 535 nm, respectively. All fluorescence measurements were corrected for background fluorescence and protein concentration. Using untreated cells as a reference, the antioxidant and prooxidant outcomes were evaluated by comparing five measurements and expressed as a percentage of untreated control cells.