DAF-FM (4-amino-5-methylamino-2′,7′-difluorofluorescein) is a cell-impermeant, fluorescent probe for detecting and quantifying low concentrations of nitric oxide (NO). DAF-FM does not need to be activated by cytosolic enzymes and is suitable to detect NO in extracellular matrix.
The fluorescence quantum yield of DAF-FM is ~0.005, but it increases about 160-fold to ~0.81 after reacting with NO and forming a fluorescent benzotriazole (excitation/emission maxima at 495/515 nm) .
The NO detection limit of DAF-FM (~3 nM) is more sensitive than that of DAF-2 (~5 nM). The fluorescence of the NO adduct of DAF-FM is independent of pH above pH 5.5. Moreover, the NO adduct of DAF-FM demonstrates a significantly enhanced photostability compared to that of DAF-2, ensuring reliable results and additional time for imaging.
DAF-FM should be dissolved in DMSO and then used to prepare a working solution. Buffers containing bovine serum albumin (BSA) or phenol red can affect the fluorescence and should be used cautiously.
The cell-permeant version of DAF FM — DAF-FM DA is also available.
