Esteras Noemí, Adjobo-Hermans Merel J W, Abramov Andrey Y, Koopman Werner J H
Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology, London, United Kingdom.
Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud Center for Mitochondrial Medicine, Radboudumc, Nijmegen, The Netherlands.
Methods Cell Biol. 2020;155:221-245. doi: 10.1016/bs.mcb.2019.10.003. Epub 2019 Dec 10.
Assessment of the mitochondrial membrane potential (Δψ) in living cells, although not trivial, can be used to estimate mitochondrial bioenergetic state. For this purpose, fluorescent lipophilic cations are broadly applied. These cations enter cells and accumulate primarily in the mitochondrial matrix in a Δψ-dependent manner. Here, we describe the use of the cations tetramethylrhodamine methyl ester (TMRM) and rhodamine 123 (Rhod123) for semi-quantitative Δψ analysis in living mammalian cells. Two different strategies are presented: (1) steady-state measurements that are suited to compare Δψ between different conditions (i.e., for comparing disease states or treatments) and (2) dynamic measurements allowing temporal monitoring of Δψ changes (i.e., to assess the effect of acute perturbations). We discuss the rationale for the use of TMRM and Rhod123 in their non-quenching/redistribution and quenching mode, how these modes are associated with different fluorescence responses, and how data can be interpreted. Practically, three experimental protocols are provided describing the use of TMRM and/or Rhod123 to assess Δψ in primary human skin fibroblasts (PHSFs) and neuron/astrocyte co-cultures by live-cell fluorescence microscopy.
评估活细胞中的线粒体膜电位（Δψ）虽非易事，但可用于估计线粒体生物能量状态。为此，广泛应用荧光亲脂性阳离子。这些阳离子进入细胞并主要以Δψ依赖的方式在线粒体基质中积累。在此，我们描述了使用阳离子四甲基罗丹明甲酯（TMRM）和罗丹明123（Rhod123）对活的哺乳动物细胞进行半定量Δψ分析。介绍了两种不同的策略：（1）稳态测量，适用于比较不同条件下的Δψ（即比较疾病状态或治疗方法）；（2）动态测量，允许对Δψ变化进行实时监测（即评估急性扰动的影响）。我们讨论了在非猝灭/重新分布和猝灭模式下使用TMRM和Rhod123的原理，这些模式如何与不同的荧光反应相关，以及如何解释数据。实际上，提供了三个实验方案，描述了通过活细胞荧光显微镜使用TMRM和/或Rhod123评估原代人皮肤成纤维细胞（PHSFs）和神经元/星形胶质细胞共培养物中Δψ的方法。
