Mitochondrial dysfunction causes dozens of debilitating diseases, and is implicated in the etiology of type 2 diabetes, Parkinson's, and Alzheimer's diseases, among others. However, development of mitochondrially targeted therapeutic agents has been impeded by the lack of high-throughput screening techniques that are capable of distinguishing in intact cells the mitochondrial membrane potential (deltapsi(m)) from the plasma membrane potential, (deltapsi(p)). We report here a fluorescence resonance energy transfer (FRET) assay that specifically monitors deltapsi(m) that is not confounded by background signal arising from potentiometric dye responding to deltapsi(p). The technique relies on energy transfer between nonyl acridine orange (NAO), which stains diphosphatidyl glycerol (cardiolipin) that is indigenous to the inner mitochondrial membrane, and tetramethylrhodamine methyl ester (TMR), a potentiometric dye that is sequestered by mitochondria as a Nernstian function of deltapsi(m) and concentration. FRET occurs only when both dyes co-localize to the mitochondria, and results in quenching of NAO emission by TMR in proportion to deltapsi(m). Validation studies using compounds with well-characterized mitochondrial effects, including oligomycin, CCCP+, bongkrekic acid, cyclosporin A, nigericin, ADP, and ruthenium red, demonstrate that the FRET-based deltapsi(m) assay responds in accord with the known pharmacology. Validation studies assessing the suitability of the technique for high-throughput compound screening indicate that the assay provides a sensitive and robust assessment not only of mitochondrial integrity in situ, but also, when used in conjunction with agents such as cyclosporin A, an indicator of permeability transition.

中文翻译：

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使用荧光共振能量转移原位高通量评估线粒体膜电位

线粒体功能障碍会导致数十种使人衰弱的疾病，并与 2 型糖尿病、帕金森病和阿尔茨海默病等疾病的病因有关。然而，由于缺乏能够区分完整细胞中线粒体膜电位 (deltapsi(m)) 和质膜电位 (deltapsi(p)) 的高通量筛选技术，线粒体靶向治疗药物的开发受到阻碍. 我们在这里报告了一种荧光共振能量转移 (FRET) 检测，它专门监测 deltapsi(m)，它不会被电位染料响应 deltapsi(p) 产生的背景信号混淆。该技术依赖于壬基吖啶橙 (NAO)、它染色线粒体内膜固有的二磷脂酰甘油（心磷脂）和四甲基罗丹明甲酯（TMR），这是一种电位染料，由线粒体作为 deltapsi(m) 和浓度的 Nernstian 函数进行隔离。FRET 仅当两种染料共定位到线粒体时才会发生，并导致 TMR 与 deltapsi(m) 成比例地淬灭 NAO 发射。使用具有充分表征的线粒体效应的化合物（包括寡霉素、CCCP+、bongkrekic 酸、环孢菌素 A、尼日利亚菌素、ADP 和钌红）进行的验证研究表明，基于 FRET 的 deltapsi(m) 测定的响应符合已知的药理学。