Reactive Oxygen Species (ROS) Assay (DCFDA Assay)

HeLa cells were seeded into dark, clear-bottom 96-well microplates at 2.5 x 10⁶ cells/ml (2.5x10⁵ cells per well). The cells were incubated at 37°C and allowed to adhere overnight. The medium was thereafter aspirated from each well, followed by rinsing with 1X buffer provided in the assay kit (Abcam, Cat. No. ab113851). The buffer was aspirated and the cells stained with 100 μl of diluted 2′,7′-dichlorofluorescein diacetate (DCFDA) solution (25 μM). Stained cells were incubated for 45 min at 37°C in the dark. After 45 min, DCFDA solution was removed, cells were rinsed with 1x buffer, the rinse buffer was removed and the cells were treated, in duplicate, with 100 μl of Compound 1 (6.25 to 100 μg/ml). The Fluorescence Intensity (FI) (Ex/Em = 485/535 nm) of each well was then read (CLARIO Star Microplate reader, BMG Labtech, UK) at 3 and 18 h following treatment. Background wells (untreated or diluent-treated stained cells), as well as blank wells (medium only), were included in each experiment. Each experiment was repeated three times. Cellular ROS data were then analysed and presented as fold changes compared to the negative control.