Using diaminofluoresceins (DAFs) in nitric oxide research

Diaminofluoresceins (DAFs) are fluorescent probes widely applied to measure nitric oxide (NO) formation in cells and tissues. The main advantages of these compounds are their availability and low cost, and the general availability of instruments able to detect green fluorescence in all laboratories; these include fluorimeters, flow cytometers, and fluorescent microscopes. What made these molecules particularly interesting for many scientists approaching the NO field is that they are apparently very easy to use, as compared with other techniques requiring specific instrumentation and knowledge like chemiluminescence and electron paramagnetic resonance. However, the reactivity and biological chemistry of these probes in the cellular environment is rather complex and still not fully understood. Moreover, secondary reactions with ascorbate, or interference with thiols occur in cells. Therefore, the use of DAFs requires specific experimental planning and a careful interpretation of the results obtained. In this methodological review, we described in detail what is known about the reactivity of DAFs, their application in biological assays, list some principles to help experimental planning, including the necessary controls, and list the caveats concerning result interpretation. These guiding principles will help to understand the "Method behind our DAF-madness".