Fluorescence-based measurement of nitric oxide synthase activity in activated rat macrophages using dichlorofluorescin

Nitric Oxide : Biology and Chemistry
A Imrich, L Kobzik

Abstract

We investigated the utility of the oxidant-reactive probe dichlorofluorescin (DCFH) for measurement of NO synthase (NOS) activity in rat alveolar macrophages (AMs) activated by culture for 18 h with interferon-gamma (IFN-gamma, 25 U/ml). Using both microplate-based fluorometry and flow cytometric analysis, AMs treated with l (but not d)-arginine showed a dose- and time-dependent increase in DCFH oxidation above buffer control (e.g., DCF production (nM): control, d-arginine 100 microM, l-arginine 100 microM respectively; 91+/-9, 76+/-11, 396+/-45, mean +/- SE, n = 6, 120 min). Furthermore, the NOS inhibitor (nitro-l-arginine) showed complete inhibition of the l-arginine-dependent DCF production. Parallel assays showed a strong correlation in DCFH oxidation with nitrite production in the same samples (e.g., DCF production (nM): 143, 222, 409; nitrite (microM): 2.2, 3.6, 7.1; for 5, 10, 50 microM l-arginine, respectively). In contrast, the alternate oxidant-reactive probes hydroethidine (HE) and dihydrorhodamine (DHR) did not report increased oxidant production in activated AMs incubated with l-arginine, despite their ability to easily detect intracellular superoxide anion production in cells treated with menadione (100 microM). W...Continue Reading

References

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Citations

Oct 17, 2008·Molecular Biology of the Cell·Beata Wojciak-StothardJames M Leiper
Apr 2, 2008·Biological & Pharmaceutical Bulletin·Shvetambri AroraVirinder Singh Parmar
Jun 11, 2002·Journal of Molecular and Cellular Cardiology·Nanette H Bishopric
Sep 12, 2002·Nitric Oxide : Biology and Chemistry·Prashant SharmaMadhu Dikshit
Jul 13, 2000·Journal of Chromatography. B, Biomedical Sciences and Applications·D TsikasJ C Frölich
Nov 22, 2011·Nitric Oxide : Biology and Chemistry·Ajit KumarHanumantharao G Raj
Jun 16, 2009·Free Radical Biology & Medicine·Hao HongWeibo Cai
Sep 5, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Dimitrios Tsikas
Jul 27, 2015·Fish & Shellfish Immunology·Danielle Ferraz MelloAlcir Luiz Dafre
Mar 6, 2003·The Journal of Clinical Investigation·Hideo IchimuraJahar Bhattacharya
Mar 23, 2011·Nitric Oxide : Biology and Chemistry·Pinar UlkerOguz K Baskurt
Feb 3, 2009·Biological & Pharmaceutical Bulletin·Shvetambri AroraVirinder Singh Parmar
Sep 4, 2004·American Journal of Physiology. Heart and Circulatory Physiology·Ossama A HatoumDavid D Gutterman
Nov 14, 2003·American Journal of Physiology. Heart and Circulatory Physiology·Atsushi SatoDavid D Gutterman

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