Direct intracellular nitric oxide detection in isolated adult cardiomyocytes: Flow cytometric analysis using the fluorescent probe, diaminofluorescein
dc.contributor.author | Strijdom H. | |
dc.contributor.author | Muller C. | |
dc.contributor.author | Lochner A. | |
dc.date.accessioned | 2011-05-15T15:59:43Z | |
dc.date.available | 2011-05-15T15:59:43Z | |
dc.date.issued | 2004 | |
dc.description.abstract | We 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.version | Article | |
dc.identifier.citation | Journal of Molecular and Cellular Cardiology | |
dc.identifier.citation | 37 | |
dc.identifier.citation | 4 | |
dc.identifier.issn | 222828 | |
dc.identifier.other | 10.1016/j.yjmcc.2004.05.018 | |
dc.identifier.uri | http://hdl.handle.net/10019.1/11326 | |
dc.subject | 2 (n,n diethylamino)diazenolate 2 oxide | |
dc.subject | bovine serum albumin | |
dc.subject | collagenase | |
dc.subject | diaminofluorescein 2 diacetate | |
dc.subject | fluorescein derivative | |
dc.subject | n(g) nitroarginine methyl ester | |
dc.subject | nitric oxide | |
dc.subject | nitric oxide donor | |
dc.subject | nitric oxide synthase inhibitor | |
dc.subject | unclassified drug | |
dc.subject | animal cell | |
dc.subject | article | |
dc.subject | cell culture | |
dc.subject | cell isolation | |
dc.subject | controlled study | |
dc.subject | data analysis | |
dc.subject | flow cytometry | |
dc.subject | fluorescence activated cell sorting | |
dc.subject | fluorescence analysis | |
dc.subject | heart muscle cell | |
dc.subject | heart muscle ischemia | |
dc.subject | heart muscle perfusion | |
dc.subject | hypoxia | |
dc.subject | nonhuman | |
dc.subject | priority journal | |
dc.subject | rat | |
dc.subject | reproducibility | |
dc.subject | response time | |
dc.subject | statistical significance | |
dc.subject | urea cycle | |
dc.subject | Animals | |
dc.subject | Cell Hypoxia | |
dc.subject | Cells, Cultured | |
dc.subject | Flow Cytometry | |
dc.subject | Fluorescein | |
dc.subject | Fluorescent Dyes | |
dc.subject | Myocytes, Cardiac | |
dc.subject | Nitric Oxide | |
dc.subject | Nitric Oxide Synthase | |
dc.subject | Rats | |
dc.subject | Bovinae | |
dc.title | Direct intracellular nitric oxide detection in isolated adult cardiomyocytes: Flow cytometric analysis using the fluorescent probe, diaminofluorescein | |
dc.type | Article |