Flow cytometry membrane potential

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Ordonez, J. V. Wehman, N. M. Amphotericin B susceptibility of Candida species assessed by rapid flow cytometric membrane potential assay. Cytometry 1995, 22, 154-157. 

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Fig. 7.4 (a) Dose-response of the lamellarin M-induced mitochondrial depolarization in P388 cells, (b) Monitoring of the mitochondrial membrane potential (A rm) by realtime flow cytometry, using functional mitochondria isolated from P388 cells and the fluorescent probe JC-1. 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

Fig. 4. Combined use of flow cytometry/cell sorting and confocal laser scanning microscopy. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine and then analyzed for mitochondrial membrane potential and NAD(P)H fluorescence (A). Cells in different regions of the cytogram were then sorted, and subsequently stained with the DNA fluorochrome Hoechst 33342. These cells were then examined by CLSM. (B), (C), and (D) show three-dimensional reconstructions of nuclei from cells sorted from healthy, early apoptotic, and late apoptotic populations. Whereas healthy cells show normal round nuclei, early and late apoptotic cells show progressive chromatin condensation/margination and nuclear fragmentation.

The membrane potential of individual cells can be monitored with a fluorescence microscope. For this purpose, however, it is preferable to use a permeable redistribution dye with spectral characteristics that have minimal environmental sensitivity. Thus, the fluorescence intensity will reflect the degree of Nemstian accumulation of dye only and can, therefore, be readily interpreted. The plasma membrane potential can be distinguished from the organelle membrane by simply using the microscope to identify appropriate regions of the cell (44). Rhodamine-123 (Chart III) was introduced as a mitochondrial stain by Chen and co-workers (45-47) it has been used largely in qualitative studies of mitochondrial membrane potential and has been especially effective in flow cytometry applications. 

Laflamme, C. Ho, J. Veillette, M. Latremoille, M. C. Verreault, D. Meriaux, A. Duchaine, C. Flow cytometry analysis of germinating Bacillus spores, using membrane potential dye. Arch. Microbiol 2005, 183, 107-112. 

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Kalbacova, M. Vrbacky, M. Drahota, Z. Melkova, Z. Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in 2 different cell lines using flow cytometry and spectrofluorometry. Cytometry 2003, 52A, 110-116. 

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Brewis,I.A. Morton,I.E. Mohammad,S.N. Browes, C. E. Moore, H. D. M. Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry. J. Androl. 2000, 21, 238-249. 

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