Open Access

Application of 4,5-diaminofluorescein to reliably measure nitric oxide released from endothelial cellsin vitro

  • Thomas R. Räthel1,
  • Jürgen F. Leikert1,
  • Angelika M. Vollmar1 and
  • Verena M. Dirsch1Email author
Biological Procedures Online5:51136

Received: 25 February 2003

Accepted: 19 May 2003


Here we describe in more depth the previously published application of the fluorescent probe 4,5-diaminofluorescein (DAF-2) in order to reliably measure low levels of nitric oxide (NO) as released from human endothelial cells invitro. The used approach is based on the following considerations a) use low concentrations of DAF-2 (0.1 µM) in order to reduce the contribution of DAF-2 auto-fluorescence to the measured total fluorescence, and b) subtract the DAF-2 auto-fluorescence from the measured total fluorescence. The advantage of this method is the reliable quantification of NO in a biological system in the nanomolar range once thoroughly validated. Here we focus in addition to the previous publication (Leikertet al.,FEBS Lett 2001, 506:131–134) on aspects of validation procedures as well as limitations and pitfalls of this method.

Indexing terms

Investigative TechniquesBiological AssayNitric-Oxide Synthase