Direct intracellular nitric oxide detection in isolated adult cardiomyocytes: Flow cytometric analysis using the fluorescent probe, diaminofluorescein

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dc.contributor.authorStrijdom H.
dc.contributor.authorMuller C.
dc.contributor.authorLochner A.
dc.date.accessioned2011-05-15T15:59:43Z
dc.date.available2011-05-15T15:59:43Z
dc.date.issued2004
dc.description.abstractWe assessed the possibility to detect intracellular nitric oxide (NO) with the NO-specific probe 4,5-diaminofluorescein-2/diacetate (DAF-2/DA), by flow cytometry, in fresh adult rat cardiomyocytes, and compared the findings with results obtained from quantitation of cellular nitrate/nitrite (NOx) levels. Methods. - Cardiomyocytes were isolated by collagenase perfusion, followed by incubation in a Krebs-Henseleit/2% bovine serum albumin buffer in the presence of 10 μM DAF-2/DA (∼0.5 × 106 cells/ml). Experimental conditions were: (i) baseline control, (ii) NO donor (2-(N,N-diethylamino)-diazenolate 2-oxide, DEA/NO) administration, and (iii) 120 min simulated ischemia (hypoxia). In addition, control and hypoxic groups were incubated with the NO synthase (NOS) inhibitor, NW-nitro-L-arginine methyl ester (L-NAME). Following incubation and washing, intracellular fluorescence of DAF-triazol (DAF-2T, oxidized form of DAF-2/DA) was analyzed by flow cytometry. NOx levels were determined with an NOx assay. Fluorescence-activated cell sorter (FACS) data were expressed as mean fluorescence intensity (percentage of control) and NOx levels as pmol/106 cells. Results. - Optimal baseline fluorescence was obtained when myocytes were incubated with DAF-2/DA for 3 h at 37 °C. The NO donor DEA/NO (500 μM) and hypoxia significantly increased DAF fluorescence and NOx levels. L-NAME addition significantly reversed these trends in the hypoxia groups. Conclusions. - We have demonstrated that intracellular NO can be detected in fresh isolated adult cardiomyocytes by flow cytometry with 10 μM DAF-2/DA. Furthermore, we demonstrated that hypoxia is an activator of adult cardiomyocyte NOS, as demonstrated by both end-points. Reproducibility observed between results obtained by FACS analysis and NOx assays suggests that DAF-2/DA fluorescence can be regarded as an independent marker for intracellular NO in cardiomyocytes. © 2004 Elsevier Ltd. All rights reserved.
dc.description.versionArticle
dc.identifier.citationJournal of Molecular and Cellular Cardiology
dc.identifier.citation37
dc.identifier.citation4
dc.identifier.issn222828
dc.identifier.other10.1016/j.yjmcc.2004.05.018
dc.identifier.urihttp://hdl.handle.net/10019.1/11326
dc.subject2 (n,n diethylamino)diazenolate 2 oxide
dc.subjectbovine serum albumin
dc.subjectcollagenase
dc.subjectdiaminofluorescein 2 diacetate
dc.subjectfluorescein derivative
dc.subjectn(g) nitroarginine methyl ester
dc.subjectnitric oxide
dc.subjectnitric oxide donor
dc.subjectnitric oxide synthase inhibitor
dc.subjectunclassified drug
dc.subjectanimal cell
dc.subjectarticle
dc.subjectcell culture
dc.subjectcell isolation
dc.subjectcontrolled study
dc.subjectdata analysis
dc.subjectflow cytometry
dc.subjectfluorescence activated cell sorting
dc.subjectfluorescence analysis
dc.subjectheart muscle cell
dc.subjectheart muscle ischemia
dc.subjectheart muscle perfusion
dc.subjecthypoxia
dc.subjectnonhuman
dc.subjectpriority journal
dc.subjectrat
dc.subjectreproducibility
dc.subjectresponse time
dc.subjectstatistical significance
dc.subjecturea cycle
dc.subjectAnimals
dc.subjectCell Hypoxia
dc.subjectCells, Cultured
dc.subjectFlow Cytometry
dc.subjectFluorescein
dc.subjectFluorescent Dyes
dc.subjectMyocytes, Cardiac
dc.subjectNitric Oxide
dc.subjectNitric Oxide Synthase
dc.subjectRats
dc.subjectBovinae
dc.titleDirect intracellular nitric oxide detection in isolated adult cardiomyocytes: Flow cytometric analysis using the fluorescent probe, diaminofluorescein
dc.typeArticle
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