Supplementary Material for: Characterization of a Fluorescent Probe for Imaging Nitric Oxide

<b><i>Background:</i></b> Nitric oxide (NO), a potent vasodilator and anti-atherogenic molecule, is synthesized in various cell types, including vascular endothelial cells (ECs). The biological importance of NO enforces the need to develop and characterize specific and sensitive probes. To date, several fluorophores, chromophores and colorimetric techniques have been developed to detect NO or its metabolites (NO₂ and NO₃) in biological fluids, viable cells or cell lysates. <b><i>Methods:</i></b> Recently, a novel probe (NO₅₅₀) has been developed and reported to detect NO in solutions and in primary astrocytes and neuronal cells with a fluorescence signal arising from a nonfluorescent background. <b><i>Results:</i></b> Here, we report further characterization of this probe by optimizing conditions for the detection and imaging of NO products in primary vascular ECs, fibroblasts, and embryonic stem cell- and induced pluripotent stem cell-derived ECs in the absence and presence of pharmacological agents that modulate NO levels. In addition, we studied the stability of this probe in cells over time and evaluated its compartmentalization in reference to organelle-labeling dyes. Finally, we synthesized an inherently fluorescent diazo ring compound (AZO₅₅₀) that is expected to form when the nonfluorescent NO₅₅₀ reacts with cellular NO, and compared its cellular distribution with that of NO₅₅₀. <b><i>Conclusion:</i></b> NO₅₅₀ is a promising agent for imaging NO at baseline and in response to pharmacological agents that modulate its levels