On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide

Free Radic Biol Med. 2005 Aug 1;39(3):327-35. doi: 10.1016/j.freeradbiomed.2005.03.017. Epub 2005 Apr 9.


The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Artifacts
  • Cells, Cultured
  • Endothelial Cells / metabolism*
  • Fluoresceins* / chemistry
  • Humans
  • In Vitro Techniques
  • Indicators and Reagents / chemistry
  • Nitric Oxide / analysis*
  • Oxidation-Reduction
  • Peroxynitrous Acid / chemistry
  • Sensitivity and Specificity


  • 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate
  • Fluoresceins
  • Indicators and Reagents
  • Peroxynitrous Acid
  • Nitric Oxide