RAW264.7 Cell Nitric Oxide Production

The RAW264.7 cell was seeded at seeding concentration 1 × 10⁴ cells/well. At first, the sample was pretreated with different concentrations [20 (link)]. After 1 h, the cell was stimulated with LPS (1 μg/mL) collected from (Sigma-Aldrich, St. Louis, MO, USA). The cell supernatant was collected after 24 h of incubation. NO (Nitric oxide) production was measured using Griess reagent by colorimetric Griess reaction (100 mL of pre-mixed Griess reagent from Sigma-Aldrich, St. Louis, MO, USA). The equal volume of reagent was added with supernatant and incubated for 10 min to determine NO production. Two multimode microplate readers were used to measure the absorbance at 450 nm for Nitric oxide production assay.