Griess reagent assay

Isolated endothelial cells were seeded at 3 × 10⁵ cells/mL into 6 cm cell culture plates and allowed to adhere at 37°C and 5% CO₂ overnight. After 24 hr a Griess Assay was performed (nitrate/nitrite colorimetric assay, Cayman chemical) according to the manufacturer’s instruction. Once complete, the cells were lysed in 1% (wt/vol) NP 40, 25 mM Tris–HCl pH 7.6, 0.1% SDS, 0.15 M NaCl, 1% sodium deoxycholate, and 1× Protease Inhibitor Cocktail (Roche), and concentration of nitrate/nitrite was normalized against protein concentration, measured using the BCA protein detection kit (ThermoFisher).