A flow-cytometry assisted segregation of responding and non-responding population of endothelial cells for enhanced detection of intracellular nitric oxide production.

Nitric oxide (NO) is an important paracrine substance released by the endothelium to regulate vasomotor tone. The constitutive levels of endothelium dependent NO production is low. However, it is induced significantly in response to certain environmental and biological stimuli. An accurate evaluation of such stimulus induced NO release is of pharmacological significance. We observed that the sensitivity of NO detection in endothelial cells is compromised by baseline fluorescence emanated from non-activated cells resulting in ambiguous detection. In order to measure NO levels in activated population independent of non-activated cells, we segregated DAF-FM loaded cells based on their fluorescence intensity using flow-cytometry. Specific agonists like bradykinin, VEGF and insulin enhanced the proportion of activated cells. This effect was partially blocked in presence of NO synthase inhibitor, N(G)-nitro-L-arginine-methyl ester (L-NAME). We demonstrate that the fluorescence yield of activated population serves as a sensitive measure to evaluate agonist induced nitric oxide production in endothelial cells. Such increase in NO production in activated cells was also associated with increased eNOS phosphorylation at Ser-1177. While the endothelial cells showed heterogeneity with respect to NO production, immuno-phenotyping for endothelial cell-surface markers revealed a homogenous population.