Measurement of NO production

RAW 264.7 cells seeded in 96-well plate at a density of 5 × 10⁴ cells/100 μL per well were treated with PSE for 4 h, rinsed with PBS, and then treated with 1 μg/mL of LPS. After 20 h, 50 μL of supernatant was harvested and mixed with 50 μL of Griess reagent (.1% naphthylethylenediamine and 1% sulfanilamide in 5% phosphoric acid solution). The optical density at 540 nm was measured after 5 min. The production of NO was quantified based on NaNO₂ (Junsei Chemical, Chuo-ku, Tokyo, Japan) standard reference curve.