Measurement of intracellular ROS

Intracellular reactive oxygen species (ROS) was measured using a Cellular ROS Detection Assay Kit (Cat#: ab113851, Abcam, Cambridge, UK). The kit contains 2′, 7′-Dichlorofluorescin diacetate (DCFDA), a cell permeable fluorogenic dye to measure hydroxyl, peroxyl and other ROS activity within the cell, according to the manufacturer's protocol. Briefly, cells were seeded in 96-well transparent bottom black-plate. After drug treatment for 24 h, the media was removed and 20 μM DCFDA was added to the corresponding wells and incubated for 30 min at 37 °C. The dye was washed away before reading. The signal was detected by the fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm, respectively (Multimode microplate readers TECAN 200/200Pro, TECAN Group Ltd., Männedorf, Switzerland). Average relative fluorescence of control was equated to 100%, with treatment conditions calculated proportionally and signal was corrected by background signal and adjusted to the cell viability.