*3.11. Nitrite Production Measurement*

The concentration of nitrite in the cultured media was used as a measure of NO production. The assay was performed as previously described [30]. RAW 264.7 cells were plated at a density of 5 <sup>×</sup> 10<sup>5</sup> per well in a 24-well culture plate and incubated in a 37 ◦C humidified incubator with 5% CO2 in air for 18 h. The incubated cells were pretreated with phenol-red-free medium containing various concentrations of tested compounds for 30 min, followed by 1 μg/mL of LPS treatment for 18 h more. Aliquots of the supernatant from each well (100 μL) were transferred to a 96-well plate, and nitrite concentration was measured using Griess reagent. After the Griess reaction, MTT solution (final concentration of 500 μg/mL) was added to each well and further incubated for 4 h at 37 ◦C. Each medium was discarded, and dimethyl sulfoxide was added to each well to dissolve generated formazan. The absorbance was measured at 570 nm, and percent survival was determined by comparison with LPS treated control group. All reagents used in the nitrite production measurement were purchased from Sigma-Aldrich (St. Louis, MO, USA).
