A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope.
A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope.
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一种使用荧光微珠和倒置显微镜尤斯型腔室监测细胞高度变化的简单方法。
DOI：
10.1007/bf00371117
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Wills,NK
中科院分区：
文献类型：
--
作者：
Crowe,WE;Wills,NK
在这项研究中，我们报告了培养上皮细胞离子转运研究的两个进展。首先，提出了一种简便的测量细胞表观高度的方法，该方法使用荧光微珠作为根尖和基底细胞表面的高对比度标志。然后，表观细胞高度被用作监测细胞体积因渗透压扰动而变化的时间进程的指示器。其次，介绍了一种用于倒置荧光显微镜的Ussing类型的小室设计，该小室可用于跨上皮电性质的测定。使用这两种方法，我们获得了培养的肾(A6)上皮细胞高度和跨上皮电参数的同时测量。细胞高度的测量是通过交替聚焦标记根尖和基底表面的微珠之间的显微镜来测量的。这两个表面之间的距离是通过电位器的电压输出来测量的，电位器机械地连接到显微镜的精细聚焦旋钮上。随着浴液渗透压的降低，细胞高度和跨上皮Na+转运速率(以短路电流衡量)增加。细胞高度的增加比ISC的变化早几分钟，这表明细胞体积的变化与跨上皮Na+转运之间缺乏直接联系。荧光微珠细胞高度法和Ussing-type小室都可以与膜片钳技术、细胞内微电极穿刺法或细胞内成分的荧光探针结合使用。因此，该系统可能有利于上皮细胞体积和通道调节的研究。
In this study, we report two developments for studies of ion transport in cultured epithelial cells. First, a convenient method is presented for measuring apparent cell height using fluorescent microbeads as high-contrast landmarks of the apical and basal cell surfaces. The apparent cell height is then used as an indicator to monitor the time course of changes in cell volume in response to osmotic perturbations. Second, an Ussing-type chamber design for the inverted fluorescence microscope is presented, which allows determination of transepithelial electrical properties. Using these two methods, we obtained simultaneous measurements of cell height and transepithelial electrical parameters for cultured renal (A6) epithelium. Cell height was measured by alternately focusing the microscope between microbeads marking the apical and basal surfaces. The distance between these two surfaces was measured electrically from the voltage output of a potentiometer that was mechanically coupled to the fine-focusing knob of the microscope. Following decreases in the bathing solution osmolality, the cell height and transepithelial Na+transport rate (measured as short-circuit current,ISC) increased. The increase in cell height preceded changes inISCby several minutes, suggesting a lack of direct linkage between changes in cell volume and transepithelial Na+transport. Both the fluorescent microbead cell height method and the Ussing-type chamber can be used in conjunction with patch-clamp techniques, intracellular microelectrode impalements, or fluorescent probes of intracellular composition. Therefore, this system may be advantageous for studies of epithelial cell volume and channel regulation.