Luxardi Guillaume, Reid Brian, Ferreira Fernando, Maillard Pauline, Zhao Min
Department of Dermatology, Institute for Regenerative Cures, University of California, Davis;
Department of Dermatology, Institute for Regenerative Cures, University of California, Davis.
J Vis Exp. 2015 May 3(99):e52782. doi: 10.3791/52782.
Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick's law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.
来自动物、植物和单细胞的细胞被称为细胞膜的屏障所包围，该屏障将细胞质与外界分隔开来。上皮等细胞层也形成了一道屏障，将多细胞生物的内部与外部或不同隔室分隔开来。这些屏障的一个关键特征是离子在细胞膜或细胞层上的差异分布。有两个特性使得这种分布成为可能：1）膜和上皮对特定离子具有选择性通透性；2）离子通过泵跨细胞膜和细胞层进行运输。这些特性在维持组织生理方面起着至关重要的作用，并在损伤后、修复过程中或病理状态下作为信号线索发挥作用。离子选择性自参比微电极能够在单细胞和组织水平上测量特定离子（如钙、钾或钠）的通量。该微电极包含一种离子载体混合物，它对特定离子具有选择性通透性。内部填充溶液含有一定浓度的目标离子。微电极的电势由离子的外部浓度决定。随着离子浓度的变化，微电极的电势会根据离子活度的对数而变化。当微电极在离子源或离子阱附近来回移动时（即由于离子通量而处于浓度梯度中），微电极电势会以与离子通量/梯度成比例的幅度波动。放大器放大微电极信号，并将输出记录在计算机上。然后可以使用菲克扩散定律，根据电极电势波动、微电极的偏移以及其他参数（如特定离子迁移率）来计算离子通量。在本文中，我们详细描述了使用离子选择性自参比微电极测量细胞外离子通量的方法，并展示了一些代表性结果。
