carbocyanines and mitotracker-orange

Topics: Benzimidazoles; Carbocyanines; Cell Culture Techniques; Fibroblasts; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Membrane Potential, Mitochondrial; Microscopy, Confocal; Mitochondria; Proof of Concept Study; Schizophrenia; Xanthenes

The magnitude of the inner mitochondrial membrane potential (deltapsim) appears to influence the level of certain mitochondrial activities including regulation of ionic fluxes and ATP liberation, activities that are often compartmentalized or location dependent in cells. Recent evidence suggests that within cells, mitochondria can be heterogeneous with respect to deltapsim, and that high-polarized mitochondria (high deltapsim) may occur in the subplasmalemmal cytoplasm where intercellular contact is absent. Here, we investigated whether deltapsim in oocytes and preimplantation embryos was heterogeneous and cell contact-associated.. Mouse and human oocytes and preimplantation stage embryos stained with mitochondria-specific probes rhodamine 123, MitoTracker Orange, and the deltapsim-sensitive probe JC-1, (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazoylcarbocyanine iodide), were examined by epifluorescence, scanning laser confocal, and transmission electron microscopy. The possibility that intercellular contact and deltapsim are associated was examined for oocytes, where transzonal coronal cell contacts were terminated naturally or experimentally, and for intact, disaggregated, and reconstructed cleavage stage mouse embryos.. For both oocytes and embryos, clusters of apparently high-polarized mitochondria occur in the pericortical cytoplasm in regions free from intercellular contact.. The findings suggest that mitochondria in oocytes and preimplantation embryos may be heterogeneous with respect to deltapsim. We propose that high-polarized pericortical mitochondria may have a role in the acquisition of oocyte competence and the regulation of early developmental processes that may be associated with elevated metabolism or intracellular signalling through calcium-induced calcium release pathways.

Topics: Animals; Benzimidazoles; Blastocyst; Carbocyanines; Cell Communication; Cellular Senescence; Female; Fluorescence; Fluorescent Dyes; Humans; Intracellular Membranes; Membrane Potentials; Mice; Microscopy, Confocal; Microscopy, Electron; Mitochondria; Oocytes; Rhodamine 123; Staining and Labeling; Xanthenes

Density separated trout erythrocytes, using a discontinuous Percoll gradient, yielded three distinct subfractions (top, middle and bottom) since older cells are characterized by increasing density. Cells from each subfraction were incubated with mitochondria-specific fluorescent probe Mitotracker and JC-1 in order to assess mitochondrial mass and membrane potential by means of cytofluorimetric analysis, confocal microscopy and subsequent computer-aided image analysis allowing a detailed investigation at single cell level. Both cytofluorimetric data and image analysis revealed changes in size and redistribution of mitochondria starting from the light fraction to the bottom. In particular in young erythrocytes small mitochondria were detected localized exclusively around the nucleus in a crown-like shape, the middle fraction revealed enlarged mitochondria partially scattered throughout the cytosol, whereas the last fraction represented again mitochondria with reduced size being distinctly dispersed throughout the cytosol in the cells. Concerning membrane potential considerations, our study revealed a dramatic decrease of DeltaPsi(m) in the bottom layer cell mitochondria compared to the top and unusual membrane potential increase of a subpopulation of enlarged mitochondria. DeltapH was also investigated in the three fractions by pretreating the cells with nigericin, allowing to confirm a mitochondrial energetic impairment in older cells.

Topics: Animals; Apoptosis; Benzimidazoles; Carbocyanines; Erythrocyte Aging; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Membrane Potentials; Microscopy, Confocal; Mitochondria; Nigericin; Oncorhynchus mykiss; Xanthenes