NO assay

Peritoneal macrophages (1 × 10⁵ cells/well) in a 96-well plate were incubated in the presence of three concentrations of M. alba L. (10, 30, and 100 μg/mL) for 24 h. NO was measured by determining the concentration of its stable oxidative metabolite nitrite using a microplate assay according to a described method (10). Supernatants (100 μL) were collected and mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-(1- naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid) at room temperature for 15 min. The absorbance was read at 570 nm using a microplate reader. NaNO₂ was used as a standard.