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

Objective: Maintenance of the mitochondrial membrane potential (Δψm) is fundamental for the normal performance and survival of cells such as cardiomyocytes, that have a high energy requirement. Measurement of Δψm 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 Δψm, 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 Δψm 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 Δψm. Conclusions: We conclude that JC-1 is the optimal dye to use when measuring Δψm in cardiomyocytes. (C) 2000 Elsevier Science B.V.