*4.4. Measurement of NO Production*

The Griess test was used to determine NO production. RAW 264.7 cells were plated into a 24-well plate at a density of 6 × <sup>10</sup><sup>4</sup> cells/well. After 24 h, RAW 264.7 cells were pre-treated with 50, 100, and 150 μM of wistin for 30 min, followed by treatment with LPS (0.1 μg/mL) and further incubation for 24 h. The supernatant from each well was transferred to a 96-well plate, followed by the addition of Griess reagent in a 1:1 ratio. After 15 min, absorbance was measured at 540 nm using a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific Co.).
