Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein

FEBS Lett. 2001 Oct 5;506(2):131-4. doi: 10.1016/s0014-5793(01)02901-5.


4,5-Diaminofluorescein (DAF-2) and its membrane-permeable derivate DAF-2 diacetate are fluorescent probes that have been developed to perform real-time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF-2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF-2 is possible (a) by using low DAF-2 concentrations (0.1 microM) and (b) by subtracting the DAF-2 auto-fluorescence from the measured total fluorescence. The described method allows easy real-time detection of endothelial NO formation.

MeSH terms

  • Cell Line
  • Endothelium, Vascular / chemistry*
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects
  • Enzyme Inhibitors / pharmacology
  • Fluorescein*
  • Fluorescent Dyes
  • Humans
  • Molecular Structure
  • Nitric Oxide / analysis*
  • Spectrometry, Fluorescence
  • Tetradecanoylphorbol Acetate / pharmacology
  • Triazenes / metabolism
  • omega-N-Methylarginine / pharmacology


  • 4,5-diaminofluorescein
  • Enzyme Inhibitors
  • Fluorescent Dyes
  • Triazenes
  • NOC 9
  • omega-N-Methylarginine
  • Nitric Oxide
  • Tetradecanoylphorbol Acetate
  • Fluorescein