Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device.
Intracellular nitric oxide (NO) production in a microfluidic endothelium is detected using fluorescence microscopy. Bovine pulmonary artery endothelial cells (bPAECs) were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent fluorescent DAF-FM DA/NO adduct was measured. Solutions of bradykinin, a well-known stimulus of endothelium-derived NO, activated nitric oxide synthase (NOS) in the immobilized bPAECs. This activation was inhibited using l-nitro arginine methyl ester (L-NAME), a competitive inhibitor of NOS. Importantly, the NO production was also stimulated with adenosine triphosphate (ATP) using concentrations as low as 1 microM. Previous reports on stimulating NO production using an immobilized endothelium in microfluidic channels were limited by the requirement of ATP concentrations of at least 100 microM, a value that is not physiologically relevant. The ability to monitor NO production with ATP concentrations that are similar to in vivo levels of ATP in the microcirculation represents a major advance in the use of microfluidic technology as an in vitro model of the microcirculation.
D'Amico Oblak
Teresa
T
Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, USA.
Root
Paul
P
Spence
Dana M
DM
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D001920:Bradykinin / Q000737:chemistry / Q000494:pharmacology; D002417:Cattle; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D005453:Fluorescence; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry; D012680:Sensitivity and Specificity; D012828:Silicones / Q000737:chemistry*
D004129:Dimethylpolysiloxanes; D012828:Silicones; D009569:Nitric Oxide; C013830:baysilon; D000255:Adenosine Triphosphate; D019001:Nitric Oxide Synthase; D001920:Bradykinin; D019331:NG-Nitroarginine Methyl Ester