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

Objective: Maintenance of the mitochondrial membrane potential (Delta psi m ) is fundamental for the normal performance and survival of cells such as c ardiomyocytes, that have a high energy requirement. Measurement of Delta ps i m is therefore essential in order to develop an understanding of the mole cular mechanisms controlling cardiomyocyte function. Here We have evaluated various potentiometric dyes for their ability to detect alterations of Del ta psi m. using flow cytometry and confocal microscopy. Methods: primary cu ltures of cardiomyocytes from neonate rats were treated with mitochondrial uncouplers before or after loading with Rho123. DiOC(6)(3), CMXRos or JC-1, and then analysed by flow cytometry. Apoptotic cells were identified by li ght scatter and Annexin V staining. Results: The four potentiometric dyes t ested 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 uncou plers of mitochondrial respiration. Confocal microscopic analysis confirmed that JC-1 stained mitochondria in a potential-dependent manner. In contras t. CMXRos stained cardiomyocytes irrespective of alterations in Delta psi m . Conclusions: We conclude that JC-1 is the optimal dye to use when measuri ng Delta psi m in cardiomyocytes. (C) 2000 Published by Elsevier Science B. V. All rights reserved.