Determination of NO production by macrophage RAW 264.7 cells

The level of NO production was determined by measuring nitrite production in the CM according to the Griess reaction. Macrophage RAW 264.7 cells were incubated with the test sample at various concentrations (control 0 μg/mL). Then 50 μL of sulfanilamide were added to 50 μL of the culture supernatant in a 96-well plate and incubated at room temperature for 10 min before 50 μL of the NED solution were added and incubated for 10 min. Finally, the A_{540 nm} was measured using a microplate reader (model Multiskan GO; Thermo Fisher Scientific Inc.).