Buck Institute for Research on Aging, Novato, CA, USA.
Image Analyst Software, Novato, CA, USA.
Methods Mol Biol. 2022;2497:11-61. doi: 10.1007/978-1-0716-2309-1_2.
The mitochondrial membrane potential (ΔψM) is the major component of the bioenergetic driving force responsible for most cellular ATP produced, and it controls a host of biological processes. In intact cells, assay readouts with commonly used fluorescence ΔψM probes are distorted by factors other than ΔψM. Here, we describe a protocol to calculate both ΔψM and plasma membrane potential (ΔψP) in absolute millivolts in intact single cells, or in populations of adherent, cultured cells. Our approach generates unbiased data that allows comparison of ΔψM between cell types with different geometry and ΔψP, and to follow ΔψM in time when ΔψP fluctuates. The experimental paradigm results in fluorescence microscopy time courses using a pair of cationic and anionic probes with internal calibration points that are subsequently computationally converted to millivolts on an absolute scale. The assay is compatible with wide field, confocal or two-photon microscopy. The method given here is optimized for a multiplexed, partial 96-well microplate format to record ΔψP and ΔψM responses for three consecutive treatment additions.
线粒体膜电位（ΔψM）是产生大多数细胞内 ATP 的主要生物能量驱动力成分，它控制着许多生物过程。在完整的细胞中，常用的荧光 ΔψM 探针的检测结果会受到除 ΔψM 以外的因素的扭曲。在这里，我们描述了一种在完整的单细胞或贴壁培养细胞群体中，以毫伏为单位绝对测量 ΔψM 和质膜电位（ΔψP）的方案。我们的方法生成了无偏的数据，允许比较具有不同几何形状和 ΔψP 的细胞类型之间的 ΔψM，并在 ΔψP 波动时跟踪 ΔψM 的时间变化。该实验范例使用一对带有内部校准点的阳离子和阴离子探针产生荧光显微镜时程，随后在绝对标度上通过计算将其转换为毫伏。该测定方法与宽场、共聚焦或双光子显微镜兼容。这里给出的方法针对多路复用、部分 96 孔微孔板格式进行了优化，可记录连续三次处理添加的 ΔψP 和 ΔψM 响应。
