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Nitric Oxide (Red dye): The strategy for fluorescence-based detection of NO employs an o-phenylenediamine scaffold, which in the presence of NO and air oxidizes to the corresponding aryl triazole. The electronic differences between the electron-rich diamine and electron-poor triazole groups provide a robust switch for NO detection. A crucial feature contributing to the success of this particular diamine-based probe is its high selectivity for NO under aerated conditions, as the fluorescent triazole product is not formed by reaction with superoxide, hydrogen peroxide, or peroxynitrite. We have observed poorer selectivity with diaminofluorescein (DAF) compared with our Red NO detection reagent, noting reactivity of DAF with both NO and peroxynitrite. Our probe is designed to form an insoluble precipitate upon reaction with nitric oxide. This prevents it from leaching out of cells, a problem encountered with DAF and its reaction product.
ROS (Green dye): The oxidation of the ROS detection reagent produces a green fluorescent compound. The detection reagent reacts with a broad range of reactive oxygen species, as indicated in our product literature. Reaction with such free radicals does not generate a fluorescent free radical, simply an oxidized dye that is fluorescent. Selectivity of the assay is simply determined by testing in the presence and absence of N-acetyl cysteine (NAC), a broad-spectrum ROS inhibitor. By using other inhibitors, it is even possible to classify the type of ROS that is generating the signal. This is illustrated in our workflow diagram below, following the green column in the diagram. Our protocol involves both ROS inducer and inhibitor controls to assure investigators of the selectivity of the assay.
Superoxide (Orange dye): The oxidation of the Superoxide detection reagent produces an orange fluorescent compound. The detection reagent reacts with superoxide but not other reactive oxygen species. Reaction with superoxide does not generate a fluorescent free radical, only an oxidized dye that is fluorescent. Selectivity of the assay is simply determined by testing in the presence and absence of a superoxide generator, such as actinomycin A as well as a superoxide scavenger, such as Tiron.
