Flow cytometry mitochondrial

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. 

Mitochondrial parameters Flow cytometry and confocal microscopy Fast large cell numbers Early stages of apoptosis Multiparameter option Indirect early stage changes may not be definitive proof... 

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.

Poot, M. and Pierce, R. H. (1999) Detection of changes in mitochondrial function during apoptosis by simultaneous staining with multiple fluorescent dyes and correlated multiparameter flow cytometry. Cytometry 35(4) 311-317. 

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. 

Christensen, M.E., Jansen, E.S., Sanchez, W., Waterhouse, N.J., 2013. Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release. Methods 61, 138-145. 

Kataoka, M. Fukura, Y. Shinohara, Y Baba, Y. Analysis of mitochondrial membrane potential in the cells by microchip flow cytometry. Electrophoresis 2005, 26, 3025-3031. 

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. 

Rottenberg, H. Wu, S. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim. Biophys. Acta 1998, 1404, 393-404. 

Troiano, L. Eerraresi, R. Lugli, E. Nemes, E. Roat, E. Nasi, M. Pinti, M. Cossarizza, A. Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nat. Protoc. 2007, 2, 2719-2727. 

Troiano, L. Granata, A. R. M. Cossarizza, A. Kalashnikova, G. Bianchi, R. Pini, G. Tropea, F. Carani, C. Franceschi, C. Mitochondrial membrane potential and DNA stainability in human sperm cells a flow cytometry analysis with implications for male infertility. Exp. Cell Res. 1998, 241, 384-393. 

King, M. A. Eddaoudi, A. Davies, D. C. A comparison of three flow cytometry method for evaluating mitochondrial damage during staurosporine-induced apoptosis in Jurkat cells. Cytometry 2007, 71A, 668-674. 

At the end of treatment, HL-60 cells were stained with 40 nM 3,3 -dihexyloxacarbocyanine (DiOC6 (3), Molecular Probes, Eugene, OR) for 15 min at 37 C. Mitochondrial transmembrane potential was measured by flow cytometry. At least 10,000 cells were collected in each sample. Generation of ROS was monitored with 20 pM 2, 7 -dichlorofluorescein diacetate (DCFH-DA) (Molecular Probes, Eugene, OR) staining for 1 h at 37 C and assayed by flow cytometry. 

Figure 7. Induction of mitochondrial membrane potential loss and ROS generation in HL-60 cells. (A) Cells were treated with 30 pM carnosic acid (CA), 20 pM carnosol (CL), 100 pM rosmarinic acid (RA), or 40 pM ursolic acid (UA) for 1 hr and incubated with DiOC6(3) for the last 15 min, then analyzed by flow cytometry. The percentage was the mean value of DiOC6(3) intensity normalized to control (B) Analysis if reactive oxygen species (ROS) generation after h treatment. Cells were incubated with 20 pM DCFH-DA for the last 1 h, then analyzed by flow cytometry. The percentage was the mean value of DCF fluorescence intensity normalized to control...

Danilovich, G. V. Danilovich, Yu. V. Gorchev, V. F. Comparative study of plasma and inner mitochondrial membrane polarization in smooth muscle cell by spectrofluorimetry and flow cytometry using... 

Ludovico, R Sansonetty, K Corte-Real, M. Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry. Microbiology 2001,147, 3335-3343. 

Sureda, E X. Escubedo, E. Gabriel, C. Comas, J. Camarasa, J. Camins, A. Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry. Cytometry 1997,28,74-80. 

Salvioli, S. Maseroli, R. Razienza, T. L. Bobyleva, V. Cossarizza, A. Use of flow cytometry as a tool to study mitochondrial membrane potential in isolated, living hepatocytes. Biochemistry (Moscow) 1998,63, 235-238. 

Guthrie, H. D. Welch, G. R. Determination of high mitochondrial membrane potential in spermatozoa loaded with the mitochondrial prohe 5,5 -6,6 -tetrachloro-l, F, 3,3 -tetraethylhenzimidazo-lyl-carbocyanine iodide (JC-1) hy using fluorescence-activated flow cytometry. Methods Mol. Biol. 2008, 477, 89-97. 

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