2.5. Measurement of Reactive Oxygen Species (ROS) Production

2′, 7′-Dichlorofluorescin diacetate (DCFDA) is a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activities within the cell. This study used a DCFDA Cellular ROS Detection Assay Kit (ab113851, Abcam) to measure intracellular ROS according to the manufacturer’s protocol. DCFDA-labeled erythrocytes were treated with different concentrations of tert-butyl hydroperoxide (TBHP) (5 mM, 10 mM, 50 mM and 100 mM) at 37 °C for 30 min. TBHP mimics ROS activity to oxidize DCFDA to fluorescent DCF. Finally, the mean fluorescence intensities obtained from 50,000 erythrocytes were measured using FACSCalibur (Becton Dickinson, NJ, USA). All samples were analyzed in triplicate, and the intraassay CV was 4.1 ± 0.7%.