A novel optical assay system for the quantitative measurement of chemotaxis
A novel optical assay system for the quantitative measurement of chemotaxis
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DOI：
10.1016/j.jim.2003.07.008
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发表时间：
2003-11-01
影响因子：
2.2
通讯作者：
Kikuchi, Y
中科院分区：
文献类型：
--
作者：
Kanegasaki, S;Nomura, Y;Kikuchi, Y
我们已经开发了一种光学可接近的水平趋化装置，由蚀刻的硅衬底和平板玻璃组成，两者都形成了两个隔室，其间有一个5微米深的微孔板。该装置与带有用于注射细胞的孔的不锈钢钻头和不同隔室的化学吸引剂保持在一起。通道中细胞的迁移是使用一台ccd摄像机以时间间隔跟踪的。通过开发一种在通道边缘排列细胞的方法，我们可以成功地将趋化试验所需的细胞数量减少到100个或更少，具体取决于实验。为了防止内容物通过连通的微通道在相邻隔间之间不断流动，在对准单元后，支架顶端的空间被填充了介质。通过使用荧光探针，我们在实验上证明了可以保持稳定的浓度梯度。此外，我们确定了使用模型趋化因子和计算流体动力学程序建立的梯度的理论细节。以人中性粒细胞和IL-8为模型，获得了重复性较好的细胞迁移动力学结果。其他细胞，如嗜酸性粒细胞、嗜碱性粒细胞和Jurkat淋巴细胞也向适当的趋化因子迁移。(C)2003爱思唯尔B.V.保留所有权利。
We have developed an optically accessible, horizontal chemotaxis apparatus consisting of an etched silicon substrate and a flat glass plate, both of which form two compartments with a 5-mum-deep microchannet in between. The device is held together with a stainless steel bolder with holes for injecting cells and a chemoattractant to the different compartments. Migration of cells in the channel is traced with time-lapse intervals using a CCD camera. By developing a method for aligning cells at the edge of the channel, we could successfully reduce the number of cells required for a chemotactic assay, depending on the experiment, to 100 or less. To prevent ceaseless flow of contents between the adjacent compartments via the communicating microchannel, a space at the top end of the holder was filled with medium after aligning the cells. By using a fluorescent probe, we demonstrated experimentally that a stable concentration gradient could be maintained. Furthermore, we determined theoretical details of the gradient established using a model chemokine and a computational fluid dynamics code. Reproducible kinetic results of cell migration were obtained using human neutrophils and IL-8 as a model. Migration of other cells such as eosinophils, basophils and Jurkat lymphocytes toward the appropriate chemokines were also demonstrated. (C) 2003 Elsevier B.V. All rights reserved.