title: 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-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.
authors.affiliation: Faculty of Pharmacy, Pharmaceutical Chemistry Department, King Abdulaziz University, Jeddah 21589, Saudi Arabia. sabdulhai@kau.edu.sa.|Faculty of Pharmacy, Department of Analytical Chemistry, Zagazig University, Zagazig 44519, Egypt. sabdulhai@kau.edu.sa.
authors.forename: Soad S
authors.initials: SS
authors.lastname: Abd El-Hay
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA. colyercl@wfu.edu.
authors.forename: Christa L
authors.initials: CL
authors.lastname: Colyer
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000378:metabolism; D002021:Buffers; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000592:standards*; D057230:Limit of Detection; D008297:Male; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis* / Q000096:biosynthesis; D020030:Nitric Oxide Donors / Q000737:chemistry* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011765:Pyruvaldehyde / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D046509:Tissue Culture Techniques
chemical_list: C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; C002385:linsidomine; D011765:Pyruvaldehyde; D008981:Molsidomine; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D000109:Acetylcholine; D019331:NG-Nitroarginine Methyl Ester
keywords: DAF-FM; SIN-1; fluorescence; microplate reader; nitric oxide