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Molecules 2017, 22(1), 127;

Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader

Faculty of Pharmacy, Pharmaceutical Chemistry Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia
Faculty of Pharmacy, Department of Analytical Chemistry, Zagazig University, Zagazig 44519, Egypt
Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA
Author to whom correspondence should be addressed.
Academic Editor: Derek J. McPhee
Received: 23 November 2016 / Revised: 10 January 2017 / Accepted: 11 January 2017 / Published: 13 January 2017
(This article belongs to the Section Metabolites)
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Background: Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. Objective: By using the fluorescent probe 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. Methods: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. Results: Beer’s law was linear over a nanomolar range (1–10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10–1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, Nω-Nitro-l-arginine methyl ester (l-NAME)—the standard inhibitor of endothelial NO synthase—was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. Conclusions: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability. View Full-Text
Keywords: nitric oxide; DAF-FM; SIN-1; fluorescence; microplate reader nitric oxide; DAF-FM; SIN-1; fluorescence; microplate reader

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Abd El-Hay, S.S.; Colyer, C.L. Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader. Molecules 2017, 22, 127.

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