4.3. Dihydroethidium (DHE) Staining

ROS levels were determined using the DHE assay (Cat# ab236206, Abcam). Briefly, the cells were cultured on 4-well chamber slides for 4 h and treated with IL-18 (10 ng/mL) in the presence or absence of 5 and 10 μM roflumilast for 24 h. The cells were then incubated with prepared DHE (5 μM) reagent for 30 min at 37 °C in the dark. After capturing the fluorescent image density using a fluorescence microscope, the fluorescence density of DHE staining was calculated using ImageJ software. Briefly, we characterized the regions of interest in the images. The integrated density value (IDV) of fluorescence and total cells in the regions were recorded. Average levels of intracellular ROS = IDV/the average number of cells.