Membrane potential estimation by flow cytometry
Membrane potential estimation by flow cytometry
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DOI：
10.1006/meth.2000.1007
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发表时间：
2000-07-01
期刊：
影响因子：
4.8
通讯作者：
Shapiro, HM
中科院分区：
文献类型：
--
作者：
Shapiro, HM
膜电位（Delta Psi）由离子（如钠、钾、氯和氢）的浓度梯度产生和维持。在与许多细胞类型的发育、功能和病理学相关的表面受体介导的过程中，细胞质Δ Psi的变化通常在跨膜信号传导中起作用。当能量代谢被破坏时，线粒体Δ Psi降低，特别是在凋亡中。在缺乏线粒体的细菌中，Delta Psi反映了能量代谢的状态和细胞质膜的物理完整性。流式细胞术可用于估计真核细胞、原位线粒体、分离的线粒体和细菌中的膜电位，使用亲脂性阳离子染料如花青和罗丹明123或亲脂性阴离子染料如氧杂烯醇的较旧方法可检测Δ Psi的相对大的变化并鉴定包含细胞群的主要部分的亚群中的应答异质性。较新的比率测量技术可以精确测量Delta Psi，误差在10 mV或更小。除其他因素外，外排泵的作用、膜结构的变化以及细胞悬浮介质中蛋白质或脂质浓度的变化可能会导致细胞荧光发生变化，这可能会被误解为Delta Psi的变化。因此，用于估计和测量Δ Psi的技术通常需要仔细控制细胞和试剂浓度以及孵育时间，并且如果要提供准确的信息，则需要选择适当的对照。(C)北京大学出版社.
Membrane potential (Delta Psi) is generated and maintained by concentration gradients of ions such as sodium, potassium, chloride, and hydrogen. Changes in cytoplasmic Delta Psi in the course of surface-receptor-mediated processes related to the development, function, and pathology of many cell types often play a role in transmembrane signaling Cytoplasmic Delta Psi is also reduced to zero when the membrane is ruptured by chemical or physical agents. Mitochondrial Delta Psi is reduced when energy metabolism is disrupted, notably in apoptosis. In bacteria, which lack mitochondria, Delta Psi reflects both the state of energy metabolism and the physical integrity of the cytoplasmic membrane. Flow cytometry can be used to estimate membrane potential in eukaryotic cells, mitochondria in situ, isolated mitochondria, and bacteria, older methods, using lipophilic cationic dyes such as the cyanines and rhodamine 123 or lipophilic anionic dyes such as the oxonols can detect relatively large changes in Delta Psi and identify heterogeneity of response in subpopulations comprising substantial fractions of a cell population. Newer ratiometric techniques allow precise measurement of Delta Psi to within 10 mV or less. Among other factors, action of efflux pumps, changes in membrane structure, and changes in protein or lipid concentration in the medium in which cells are suspended can produce changes in cellular fluorescence which may be misinterpreted as changes in Delta Psi. Techniques for estimation and measurement of Delta Psi therefore typically require careful control of cell and reagent concentrations and incubation times and selection of appropriate controls if they are to provide accurate information. (C) 2000 Academic Press.