2.5. Effects of RF on NO Production in RAW 264.7 Macrophages Stimulated with LPS

The concentration of NO in culture medium was determined using a Griess reaction assay. Specifically, after incubation of cells in 96-well plates (1 × 10⁴ cells/well) for 24 h with LPS (1 µg/mL) and RF, 100 µL of supernatant from each well was collected and mixed with 100 µL of Griess reagent in a new 96-well plate. After an incubation of 15 min at room temperature, OD was determined at 540 nm with a microplate reader (Bio-Rad) [15].