*4.5. Measurement of ROS Production*

ROS production was determined using the 2',7'-dichlorofluorescin-diacetate (DCF-DA) assay. RAW 264.7 cells were plated into a 24-well plate at a density of 6 × 104 cells/well. After 24 h, RAW264.7 cells were pre-treated with 50, 100, and 150 μM wistin for 30 min, followed by LPS (0.1 μg/mL) treatment and further incubation for 24 h. The solution was completely removed from all the wells before incubation with a DCF-DA probe for 30 min at 37 ◦C. The cells were washed three times with HBSS before measuring fluorescence. The fluorescence intensity was measured at excitation and emission wavelengths of 485 nm and 530 nm, respectively, using a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific Co.). ROS in intact cells were detected with fluorescence microscopy using a Zeiss microscope (Zeiss, Jena, Germany). ROS production was further analyzed using flow cytometry (BD FACSVerse™, BD Bioscience).
