Adams Dany S, Levin Michael
Department of Biology and Center for Regenerative and Developmental Biology, Tufts University, Medford, Massachusetts 02155, USA.
Cold Spring Harb Protoc. 2012 Apr 1;2012(4):459-64. doi: 10.1101/pdb.prot067702.
Slow changes in steady-state (resting) transmembrane potential (V(mem)) of non-excitable cells often encode important instructive signals controlling differentiation, proliferation, and cell:cell communication. Probing the function of such bioelectric gradients in vivo or in culture requires the ability to track V(mem), to characterize endogenous patterns of differential potential, map out isopotential cell groups (compartments or cell fields), and confirm the results of functional perturbation of V(mem). The use of fluorescent bioelectricity reporters (FBRs) has become more common as continuing research and innovation have produced better and more options. These dyes are now used routinely for cell sorting and for studies of cultured cells. Important advantages over single cell electrode measurements are offered by dyes, including: (1) subcellular resolution, (2) the ability to monitor multicellular areas and volumes in vivo, (3) simplicity of use, (4) ability to measure moving targets, and (5) ability to measure over long time periods. Thus, FBRs are suitable for longitudinal studies of systems that change and move over time, for example, embryos. Existing protocols focus on measurements of rapid action potentials in cultured cells or neurons. This article describes a dye pair that can be used to measure resting V(mem) in cultured cells and in vivo in Xenopus laevis embryos and tadpoles (and is readily applied to other model systems, such as zebrafish, for studies of developmental bioelectricity). It is assumed that the reader is fully familiar with the process and terminology of fluorescence microscopy.
非兴奋性细胞静息跨膜电位（V(mem)）的缓慢变化通常编码着控制细胞分化、增殖以及细胞间通讯的重要指导信号。在体内或体外培养环境中探究此类生物电梯度的功能，需要具备追踪V(mem)、表征电位差异的内源性模式、绘制等电位细胞群（区室或细胞场）以及确认V(mem)功能扰动结果的能力。随着持续的研究与创新带来了更多更好的选择，荧光生物电报告分子（FBRs）的使用变得愈发普遍。这些染料如今常用于细胞分选以及培养细胞的研究。与单细胞电极测量相比，染料具有诸多重要优势，包括：（1）亚细胞分辨率；（2）在体内监测多细胞区域和体积的能力；（3）使用简便；（4）测量移动目标的能力；（5）长时间测量的能力。因此，FBRs适用于对随时间变化和移动的系统进行纵向研究，例如胚胎。现有的实验方案主要集中于测量培养细胞或神经元中的快速动作电位。本文介绍了一种染料对，可用于测量培养细胞以及非洲爪蟾胚胎和蝌蚪体内的静息V(mem)（并且很容易应用于其他模型系统，如斑马鱼，用于发育生物电研究）。假定读者对荧光显微镜的操作过程和术语非常熟悉。
