2.12. Nitric Oxide Assay in LPS-Stimulated RAW 264.7

NO concentration was determined in RAW 264.7 murine macrophages, seeded at a concentration of 1.5 × 10⁵ cells/well in 96-well plates for 24 h, and pre-treated with the extract (0.0006%, 0.002% and 0.006% p/v) or with 10 μM TPCK (positive control) for 2 h, before the incubation with 2 μg/mL LPS for 18 h. The amount of NO, converted into nitrite, was calculated by adding Griess reagent (solution of N-(1-naphthyl)ethylenediamine and sulfanilic acid, Invitrogen- Thermo Scientific, Waltham, MA, USA) and, after 30 min, the absorbance was measured at 540 nm by the multiwell-plate reader (EnVision, PerkinElmer, Waltham, MA, USA).