Nitric oxide determination

RAW264.7 macrophages were plated at 4 × 10⁵ cells/well in 6-well plates and pre-incubated with PC (1, 10, and 50 μM), EA (1, 10, and 50 μM), PPPs (1, 10, and 100 μg/mL) or vehicle (0.2% DMSO) for 1 h and then were incubated with LPS (1 μg/mL) for 24 h. The amount of nitrite in the culture media was detected by NO assay kits. The absorbance at 540 nm was measured with the NaNO₂ standard curve, and nitrite production was determined.