Measurement of nitric oxide synthase activity in murine endothelial cells

For determination of endothelial nitric oxide synthase activity, CD73−/− and WT lung endothelial cells were cultured in 96 well plates at a density 100,000 cells per well. After 24 h, nitric oxide production from murine EC was assessed using spectrofluorimeter Niric Oxide Synthase Detection System (Sigma-Aldrich, FCANOS1), according to the manufacturer’s protocol. For fluorometric NO measurement, the cell-permeable NOS-derived NO—sensitive 4,5-diaminofluorescein diacetate (DAF-2DA) was used. Diphenyleneiodonium chloride (DPI) was used as an ihibitor of inducible NOS (iNOS) at a concentration of 1 uM (as recommended in the protocol). The results are shown as relative fluorescence units (RFU) of NO concentration per mg of protein.