Kataoka Masatoshi, Fukura Yoko, Shinohara Yasuo, Baba Yoshinobu
Institute for Genome Research, The University of Tokushima, Japan.
Electrophoresis. 2005 Aug;26(15):3025-31. doi: 10.1002/elps.200410402.
The mitochondrial membrane potential (DeltaPsi(m)) is an important indicator of the energetic state of both the mitochondria and the cells. To develop a sensitive, convenient, and rapid method for the measurement of DeltaPsi(m), we carried out cell fluorescence assays using the Agilent 2100 bioanalyzer system which, unlike the conventional flow cytometry, is based on microfluidic technology employing fluorescence detection with a 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)(3)) fluorescent probe. The use of DiOC(6)(3) in the fluorometer was shown to be feasible for monitoring variations in DeltaPsi(m) in the mitochondria isolated from rat liver and treated with rotenone, succinate, ADP, and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). Flow cytometry analysis showed severe reduction of fluorescence intensity in Jurkat cells after treatment with 1.0 and 10 microM FCCP. However, fluorescence microscopy demonstrated obvious accumulation of fluorescence in the mitochondria and induction of diffuse cytoplasmic fluorescence not localized to the mitochondria in these cells. The dose response range of DiOC(6)(3) in the Agilent 2100 bioanalyzer system for yielding sufficient fluorescence intensity in the mitochondria of the cells was 20 nm-2.0 microM. Furthermore, significant reduction of fluorescence intensity in the cells stained with 2.0 microM DiOC(6)(3) was observed after treatment with 10 microM FCCP for 30 min. These results indicate that the Agilent 2100 bioanalyzer is potentially useful for monitoring DeltaPsi(m) in cell assays.
线粒体膜电位（ΔΨm）是线粒体和细胞能量状态的重要指标。为了开发一种灵敏、便捷且快速的测量ΔΨm的方法，我们使用安捷伦2100生物分析仪系统进行了细胞荧光测定，该系统不同于传统的流式细胞术，它基于微流控技术，采用3,3'-二己基氧杂羰花青碘化物（DiOC(6)(3)）荧光探针进行荧光检测。结果表明，在荧光计中使用DiOC(6)(3)监测从大鼠肝脏分离并用鱼藤酮、琥珀酸、ADP和羰基氰化物-对-三氟甲氧基苯腙（FCCP）处理的线粒体中ΔΨm的变化是可行的。流式细胞术分析显示，用1.0和10μM FCCP处理后的Jurkat细胞荧光强度严重降低。然而，荧光显微镜显示这些细胞中线粒体荧光明显积累，并诱导出非线粒体定位的弥漫性细胞质荧光。在安捷伦2100生物分析仪系统中，DiOC(6)(3)在细胞线粒体中产生足够荧光强度的剂量反应范围为20 nM - 2.0μM。此外，用10μM FCCP处理30分钟后，观察到用2.0μM DiOC(6)(3)染色的细胞荧光强度显著降低。这些结果表明，安捷伦2100生物分析仪在细胞检测中监测ΔΨm方面具有潜在用途。
