Development of high-throughput method for measurement of vascular nitric oxide generation in microplate reader

Abstract

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-20,70-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, Nw-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.