Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes.
OBJECTIVE
Maintenance of the mitochondrial membrane potential (Deltapsim) is fundamental for the normal performance and survival of cells such as cardiomyocytes, that have a high energy requirement. Measurement of Deltapsim 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 Deltapsim, 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, DiOC(6)(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 Deltapsim 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 Deltapsim.


CONCLUSIONS
We conclude that JC-1 is the optimal dye to use when measuring Deltapsim in cardiomyocytes.
Mathur
A
A
The Cell Biology Group, Centre for Vascular Biology and Medicine, Department of Medicine, University College London, Room G15, Rayne Institute, 5 University Street, London, UK.
Hong
Y
Y
Kemp
B K
BK
Barrientos
A A
AA
Erusalimsky
J D
JD
D000818:Animals; D017209:Apoptosis; D001562:Benzimidazoles; D002232:Carbocyanines; D002478:Cells, Cultured; D005069:Evaluation Studies as Topic; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000502:physiology*; D008564:Membrane Potentials; D018613:Microscopy, Confocal / Q000379:methods; D008929:Mitochondria, Heart / Q000502:physiology*; D009930:Organic Chemicals; D051381:Rats; D017207:Rats, Sprague-Dawley; D020112:Rhodamine 123
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C107472:red dye CMXRos; D020112:Rhodamine 123; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine