Section of Physiological Acoustics and Communication, Faculty of Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
Section of Physiological Acoustics and Communication, Faculty of Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.
J Neurosci Methods. 2018 Jan 1;293:310-320. doi: 10.1016/j.jneumeth.2017.10.014. Epub 2017 Oct 17.
Fluorescence membrane markers are efficient tools for visualizing the dynamics of membrane recycling processes in living cells. The outer hair cell (OHC) - a bipolar epithelial cell in the cochlea - possesses endocytic activity at both its apical and basal poles. The best visual overview of transcytosis in the OHC is achieved when the cell is isolated, so that both the apical and the basal poles are in the same focal plane to allow confocal imaging. Until now, fluorescent markers were applied to the extracellular environment of isolated OHCs without distinguishing the apical and basal poles. The drawback of that configuration is that apicobasal and basoapical vesicle traffic labelled at the opposite poles cannot be visualized independently because the same fluorescent marker has access to both poles.
A double-barrel, capillary perfusion system was developed to independently stain either one pole or both the apical and the basal poles of isolated OHCs using different types of fluorescence membrane markers.
Producing laminar fluid flow, the double-barrel perfusor allows investigation of the dynamics of apicobasal and basoapical vesicle traffic independently and/or simultaneously in the same OHC.
This method offers a unique option for investigating bidirectional vesicle traffic in bipolar epithelial cells, which is superior to other already established labelling techniques.
The double-barrel perfusion system, suitable for selectively staining a longitudinal section of the plasma membrane of an isolated bipolar epithelial cell, opens new possibilities for investigating cell labelling and intracellular vesicle traffic.
荧光膜标记物是可视化活细胞中膜循环过程动态的有效工具。外毛细胞（OHC）——耳蜗中的双极上皮细胞——在其顶端和基底极都具有内吞作用。当细胞被分离时，OHC 中的转胞吞作用可以获得最佳的视觉概述，这样顶端和基底极都在同一焦平面上，允许共聚焦成像。到目前为止，荧光标记物被应用于分离的 OHC 的细胞外环境中，而没有区分顶端和基底极。这种配置的缺点是，不能独立地可视化标记在相反极的顶端到基底和基底到顶端的囊泡运输，因为相同的荧光标记物可以到达两个极。
开发了一种双筒、毛细管灌注系统，用于使用不同类型的荧光膜标记物独立地染色分离的 OHC 的一个极或顶端和基底极。
双筒灌注器产生层流，允许在同一 OHC 中独立地和/或同时研究顶端到基底和基底到顶端的囊泡运输的动力学。
这种方法为研究双极上皮细胞的双向囊泡运输提供了一个独特的选择，优于其他已经建立的标记技术。
双筒灌注系统适用于选择性地染色分离的双极上皮细胞的质膜的一个纵向部分，为研究细胞标记和细胞内囊泡运输开辟了新的可能性。
