Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells.
BACKGROUND
The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values.


RESULTS
The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given.


CONCLUSIONS
Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.
Cassart
Dominique
D
Department of Pathology, Faculty of Veterinary Medicine, University of Liege, Sart Tilman B43, B-4000 Liège, Belgium. Dominique.Cassart@ulg.ac.be
Fett
Thomas
T
Sarlet
Michaël
M
Baise
Etienne
E
Coignoul
Freddy
F
Desmecht
Daniel
D
D000818:Animals; D001562:Benzimidazoles / Q000737:chemistry; D002232:Carbocyanines / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary*; D005456:Fluorescent Dyes / Q000737:chemistry; D006736:Horses / Q000097:blood* / Q000502:physiology; D007963:Leukocytes, Mononuclear / Q000502:physiology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D008928:Mitochondria / Q000502:physiology*; D012016:Reference Values; D015203:Reproducibility of Results
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine