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Cardiovascular Research 2000 46(1):126-138; doi:10.1016/S0008-6363(00)00002-X
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes

Anthony Mathura, Ying Honga, Barbara K. Kempb, Alberto Alvarez Barrientosc and Jorge D. Erusalimskya,*

aThe Cell Biology Group, Centre for Vascular Biology and Medicine, Department of Medicine, University College London, Room G15, Rayne Institute, 5 University Street, London WC1E 6JJ, UK
bThe Wolfson Institute for Biomedical Research, University College London, London WC1E 6AU, UK
cCentro de Citometrìa de Flujo y Microscopìa Confocal, Universidad Complutense de Madrid, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain

* Corresponding author. Tel.: +44-171-209-6613; fax: +44-171-209-6339 j.erusalimsky{at}ucl.ac.uk

Objective: Maintenance of the mitochondrial membrane potential ({Delta}{psi}m) is fundamental for the normal performance and survival of cells such as cardiomyocytes, that have a high energy requirement. Measurement of {Delta}{psi}m is therefore essential in order to develop an understanding of the molecular mechanisms controlling cardiomyocyte function. Here we have evaluated various potentiometric dyes for their ability to detect alterations of {Delta}{psi}m, using flow cytometry and confocal microscopy. Methods: Primary cultures of cardiomyocytes from neonate rats were treated with mitochondrial uncouplers before or after loading with Rho123, DiOC6(3), CMXRos or JC-1, and then analysed by flow cytometry. Apoptotic cells were identified by light scatter and Annexin V staining. Results: The four potentiometric dyes tested were able to discriminate between viable and apoptotic cells. However, only JC-1 was able to detect the collapse of {Delta}{psi}m induced by uncouplers of mitochondrial respiration. Confocal microscopic analysis confirmed that JC-1 stained mitochondria in a potential-dependent manner. In contrast, CMXRos stained cardiomyocytes irrespective of alterations in {Delta}{psi}m. Conclusions: We conclude that JC-1 is the optimal dye to use when measuring {Delta}{psi}m in cardiomyocytes.

KEYWORDS Apoptosis; Cardiomyopathy; Membrane potential; Mitochondria; Myocytes


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