A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells

被引：29

作者：

Farrelly, E ^{[1
]}

Amaral, MC ^{[1
]}

Marshall, L ^{[1
]}

Huang, SG ^{[1
]}

机构：

[1] Tularik Inc, Biol 2, San Francisco, CA 94080 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2001年 / 293卷 / 02期

关键词：

mitochondrial membrane potential; fluorescent probe; high-throughput screening; uncoupling protein 1;

D O I：

10.1006/abio.2001.5139

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3 ' -dipropylthiadicarbocyanine iodide (DISC3(5); DiSC(3)), In permeabilized cells, DISC, fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K+ and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H+ transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential. (C) 2001 Academic Press.

引用

页码：269 / 276

页数：8

共 31 条

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