Intensity-independent fluorometric detection of cellular nitric oxide release.
A new fluorescence method is introduced in which nitric oxide (NO)-derived higher-order oxygen complexes (NO(x)) are quantified at physiological pH. Detecting the fluorescence lifetime shift between 2,3-diaminonaphthalene and the NO(x)-derived protonated 2,3-naphthotriazole allows an intensity independent determination of the NO(x) concentration. The NO release from LPS and IFNgamma-stimulated murine macrophages and iNOS transfected hamster cells was quantified. The lower detection limit for NO2- was found to be 800 pmol/ml. Since the influence of static fluorescence quenching due to cellular components can be neglected, the method is applicable for clear cellular supernatants as well as turbid cellular suspensions.
Andrew
P J
PJ
Novartis Forschungsinstitut, Department Immunodermatology, Vienna, Austria.
Auer
M
M
Lindley
I J
IJ
Kauffmann
H F
HF
Kungl
A J
AJ
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004790:Enzyme Induction; D005456:Fluorescent Dyes; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D009569:Nitric Oxide / Q000032:analysis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D014162:Transfection
D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C026373:2,3-diaminonaphthalene; D009569:Nitric Oxide; D007371:Interferon-gamma; D015081:2-Naphthylamine; D019001:Nitric Oxide Synthase