4.6. Measurement of Nitric Oxide (NO) and Cytokine Production

The cells were plated at a density of 1 × 10⁵ cells/well in 96-well plates and were then stimulated with LPS (0.5 μg/mL) in various concentrations of compound for 20 h. The cell supernatants were collected for measurements of NO and cytokines, such as TNF-α and IL-6. NO was measured by the detection of its stable oxidative metabolite, nitrite. In brief, the cell supernatant (100 μL) was mixed with the same volume of Griess reagent. After reaction for 10 min at RT, the optical density was measured at 540 nm by a microplate reader (Spark10M, Tecan Science and Technology) and the nitrite concentration was determined using a sodium nitrite standard curve. The levels of TNF-α and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA) kits (BD Biosciences, Santa Clara, CA, USA) according to the manufacturer’s protocols [40]. 2-Amino-4-methylpyridine (AMP) and dexamethasone (Dex) were used as positive controls.