Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein.
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.
Leikert
J F
JF
Department of Pharmacy, Center of Drug Research, University of Munich, Butenandtstr. 5-13, D-81377, Munich, Germany.
Räthel
T R
TR
Müller
C
C
Vollmar
A M
AM
Dirsch
V M
VM
D002460:Cell Line; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019793:Fluorescein; D005456:Fluorescent Dyes; D006801:Humans; D015394:Molecular Structure; D009569:Nitric Oxide / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014226:Triazenes / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000494:pharmacology
C111205:4,5-diaminofluorescein; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D014226:Triazenes; C099612:NOC 9; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D013755:Tetradecanoylphorbol Acetate; D019793:Fluorescein