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 muM. 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 muM, 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.[1]References
- Fluorescence Monitoring of ATP-Stimulated, Endothelium-Derived Nitric Oxide Production in Channels of a Poly(dimethylsiloxane)-Based Microfluidic Device. D'Amico Oblak, T., Root, P., Spence, D.M. Anal. Chem. (2006) [Pubmed]
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