Wolff Christian, Fuks Bruno, Chatelain Pierre
Department of in vitro Pharmacology, UCB SA, Braine l'Alleud, Belgium.
J Biomol Screen. 2003 Oct;8(5):533-43. doi: 10.1177/1087057103257806.
In this study, the authors compared and evaluated 4 membrane potential probes in the same cellular assay: the oxonol dye DiBAC(4)(3), the FLIPR membrane potential (FMP) dye (Molecular Devices), and 2 novel fluorescence resonance energy transfer (FRET) dye systems from PanVera [CC2-DMPE/DiSBAC(2)(3)] and Axiom [DiSBAC(1)(3)/DiSBAC(1)(5)]. The kinetic parameters of each membrane probe were investigated in RBL-2H3 cells expressing an endogenous inward rectifier potassium channel (IRK1). The FMP dye presented the highest signal over background ratio whereas the FRET dyes from PanVera gave the fastest response. The determination of IC(50) values for 8 different channel modulators indicated a good correlation between the 4 membrane probe systems. The compound-dye interaction was evaluated in the presence of compounds at 10 muM and clearly indicated no effect on the FMP or the PanVera donor dye, whereas some major interference with the oxonol probes was observed. Using a cell permeabilization assay in the presence of gramicidin, the authors concluded that the FRET dyes from PanVera and the FMP dye are unable to measure the gramicidin-induced cell membrane hyperpolarizations. The 4 dye systems were investigated under high-throughput screening (HTS) conditions, and their respective Z' parameter was determined. The characteristics of each dye system and its potential use in HTS assays is discussed.
在本研究中，作者在同一细胞分析中比较并评估了4种膜电位探针：氧杂菁染料DiBAC(4)(3)、FLIPR膜电位（FMP）染料（分子设备公司），以及来自PanVera公司的2种新型荧光共振能量转移（FRET）染料系统[CC2-DMPE/DiSBAC(2)(3)]和来自Axiom公司的[DiSBAC(1)(3)/DiSBAC(1)(5)]。在表达内源性内向整流钾通道（IRK1）的RBL-2H3细胞中研究了每种膜探针的动力学参数。FMP染料呈现出最高的信号背景比，而来自PanVera公司的FRET染料响应最快。对8种不同通道调节剂的IC(50)值测定表明，4种膜探针系统之间具有良好的相关性。在存在10μM化合物的情况下评估了化合物与染料的相互作用，结果清楚地表明对FMP或PanVera供体染料没有影响，而观察到对氧杂菁探针有一些主要干扰。通过在短杆菌肽存在下进行细胞通透化分析，作者得出结论，来自PanVera公司的FRET染料和FMP染料无法测量短杆菌肽诱导的细胞膜超极化。在高通量筛选（HTS）条件下研究了这4种染料系统，并测定了它们各自的Z'参数。讨论了每种染料系统的特性及其在HTS分析中的潜在用途。
