Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) using flow cytometry.

Authors: Guthrie, Howard; Welch, Glenn

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: 11/30/2008
Publication Date: 12/1/2008
Citation: Guthrie, H.D., Welch, G.R. Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial probe 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) using flow cytometry.. Book Chapter. Methods in Molecular Biology, vol. 477: Advanced Protocols for Oxidative Stress I, Ed. D. Armstrong, DOI: 10.1007/978-1-60327-517-2_1, Humana Press New York, NY 07512. 2008. pp. 89-97

Interpretive Summary: A flow cytometric method was developed to identify viable, energized sperm cells with energized mitochondria using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear stain propidium iodine (PI). This flow cytometric method is described in detail here. When in contact with energized mitochondria, JC-1 forms aggregates which are fluorescent at 590 nm in response to 488 nm excitation. We found that the reactive oxygen species generator menadione reduced sperm motility and reduced the number of sperm with energized mitochondria in a dose responsive fashion that was closely correlated with the loss of motility. This technique should be applicable to any viable population of single cells whether recovered after culture, isolated from tissue, or from body fluids such as blood or semen.

Technical Abstract: A flow cytometric method was developed to identify viable, energized sperm cells with high mitochondrial inner transmembrane potential (''m), > 80-100 mV using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and the impermeant nuclear stain propidium iodine (PI). This flow cytometric method is described in detail here. When in contact with membranes possessing a high ''m, JC-1 forms aggregates (Jagg) which are fluorescent at 590 nm in response to 488 nm excitation. We found that the reactive oxygen species generator menadione reduced sperm motility and reduced ''m in a dose responsive fashion that was closely correlated with the loss of motility. This technique should be applicable to any viable population of single cells whether recovered after culture, isolated from tissue, or from body fluids such as blood or semen.