Measurement of ROS production

Cellular ROS generation was measured with the Cellular Reactive Oxygen Species Detection Assay Kit (Red Fluorescence), which was purchased from Abcam (Cambridge, UK) [23]. Cells were seeded at 1×10⁵ cells/100 μL per well in a 96-well plate. ROS Red Working Solution (100 μL) was added and incubated in a 37°C/5% CO₂ incubator for 60 min. Cells were then treated with test compounds for 60 min to induce ROS production. An increase in the fluorescence intensity at an excitation wavelength of 535 nm and an emission wavelength of 590 nm was monitored in bottom read mode.