Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device
Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device
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DOI：
10.1021/ac052066o
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发表时间：
2006-05-01
影响因子：
7.4
通讯作者：
Spence, Dana M.
中科院分区：
文献类型：
--
作者：
Oblak, Teresa D'Amico;Root, Paul;Spence, Dana M.
使用荧光显微镜检测微流体内皮细胞内一氧化氮（NO）的产生。牛肺动脉内皮细胞（bPAECs）负载有荧光探针二氨基二氟荧光素二乙酸酯（DAF-FM DA），并测量随后的荧光DAF-FM DA/NO加合物。缓激肽，一个众所周知的刺激内皮源性NO，激活一氧化氮合酶（NOS）在固定的bPAECs的解决方案。这种激活被抑制使用L-硝基精氨酸甲酯（L-NAME），一氧化氮合酶的竞争性抑制剂。重要的是，还用浓度低至1 μ M的三磷酸腺苷（ATP）刺激NO产生。先前关于在微流体通道中使用固定化内皮刺激NO产生的报道受限于ATP浓度至少为100 μ M的要求，该值与生理学无关。监测NO产生的ATP浓度与微循环中ATP的体内水平相似的能力代表了使用微流体技术作为微循环体外模型的重大进展。
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 mu M. 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 PM, 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.