A multilevel analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

► DAF-FM is a fluorescent probe for detection of intracellular NO formation. ► Use, virtues, and pitfalls associated with NO detection by DAF-FM are examined. ► Reaction products have been characterized by HPLC, NMR, and mass spectrometry. ► Robust, step-by-step protocols are provided for quantification of DAF-FM-T. ► Analytical separation techniques afford unequivocal identification of DAF-FM-T.