2.6. Measurement of Nitric Oxide (NO) Production

NO concentration in the cell culture medium was determined using a colorimetric assay based on the Griess reaction. To measure NO secretion, RAW 264.7 cells were seeded in 96 well plates, 5 × 10⁴ cells per well. After 24 h, cells were pretreated with different concentrations of HPAC for 2 h and then stimulated with 1 mg/mL of LPS for an additional 24 h. After LPS stimulation, a cell culture medium was collected to measure the amount of NO using the Griess reagent. In brief, 50 μL of Griess reagent was added to an equal volume of cell culture medium in a 96-well plate and the plate was smoothly agitated for 10 min on a shaker at room temperature. Finally, the concentration of nitrite was calculated from a standard curve of known concentrations of sodium nitrite dissolved in DMEM.