2.7. Measurement of Nitric Oxide (NO) Production

RAW264.7 cells (3 × 10⁵ cells/well) were pretreated with TCS (1, 10, and 100 μM) for 3 h, followed either by no stimulation or stimulation with 100 μg/mL of lipopolysaccharide (LPS). After a 24 h incubation, the supernatants were collected to test for NO. To measure nitrite, an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphtylethyenediamine dihydrochloride in 2.5% phosphoric acid) was mixed with cell culture supernatant at room temperature for 10 min. Nitrite concentration was determined and calculated using NaNO₂ as a standard solution by measuring absorbance at wavelength 540 nm using a VersaMax™ microplate reader.