4.9. Nitric Oxide Assay

RAW264.7 macrophages were cultured at a density of 2 × 10⁵ cells/mL in 96-well plates for 24 h. The cells were then pre-treated with (−)cis-khellactone (at concentrations of 50 and 100 µM), and after 2 h, stimulated with 1 µg/mL of LPS for 24 h. Following incubation, 100 µL each of supernatant and Griess reagent were added to the 96-well plates. Absorbance was then measured at 450 nm using a spectrophotometer (Tecan Group, Ltd., Mannedorf, Switzerland).