DCFDA Assay for Cellular ROS Detection

The DCFDA Cellular ROS Detection Assay kit (Abcam, Cambridge, UK) was used to detect the levels of Reactive Oxygen Species (ROS) in the cells. KG-1 cells were incubated with the cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), and then plated in duplicates in a 96-well plate and treated with either increasing concentrations of ASEE (19–114 μg/mL), or with a combination of 75 μM of Tert-Butyl Hydrogen Peroxide (TBHP) with increasing concentrations of the ASEE (control cells were treated with RPMI media). TBHP is a potent ROS inducer inside the cells, so it was used as a positive control. H₂DCFDA (the chemically reduced form of fluorescein) acts as an indicator for ROS in cells upon its oxidative conversion to the highly fluorescent 2′,7′-dichlorofluorescein (DCF), which was quantified by fluorescent spectroscopy on the Varioskan™ LUX multimode microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).