NO concentration was measured as the stable oxidized metabolite and nitrite (NO2−) using a Griess reaction kit (Promega UK Ltd, Southampton, UK). The limit of detection (LOD) was 2.5 μM (125 pmol). Manufacturer's instructions were followed. Briefly, 50 μL of sample and 50 μL of sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) were incubated at room temperature and protected from light for 5–10 minutes. Subsequently, 50 μL of 0.1% N-1-naphthylethylenediamine dihydrochloride in water was added, reincubated, and protected from light for a further 5–10 minutes. The absorbance was read at 540 nm and compared to a known standard.
The nitric oxide inhibitor NG-monomethyl-L-arginine (L-NMMA) (Sigma, Gillingham, UK) was used at a concentration of 4 mM and added after stimulation and remained for the duration of the assay.
The nitric oxide inhibitor NG-monomethyl-L-arginine (L-NMMA) (Sigma, Gillingham, UK) was used at a concentration of 4 mM and added after stimulation and remained for the duration of the assay.