Mitochondrial assays membrane potential

Intracellular Enzymes Stem Cell Isolation Cellular Membrane Potential Mitochondrial Membrane Potential Gene Reporter Assay Gene Silencing (siRNA)... 

Dye oxidation (e.g., tetrazolium reductase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, MTT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt, WST-1 3- (4,5 -carboxymethoxyphenyl) -2-(4-sulfophenyl)-2 H-tetra-zolium, MTS 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt, XTT 2,2 -di-p-nitrophenyl-5,5 -diphenyl-3,3 -(3,3 -dimethoxy-4,4 -diphe-nylenej-ditetrazolium chloride, NET), Alamar blue assays, ATP concentration (e.g., luciferase assay), oxygen consumption (e.g., oxygen electrodes, phosphorescent oxygen-sensitive dyes), mitochondrial protein and nucleic acid synthesis mitochondrial mass (e.g., mitotracker dyes) mitochondrial membrane potential (e.g., tetramethylrho-damine methyl ester, TMRM tetramethylrhodamine ethyl ester, TMRE)... 

Fig. 2. Cytogram of a combined assay for NAD(P)H levels (UV-excited blue autofluorescence) and mitochondrial membrane potential (CMXRosamine/ MitoTracker Green FM fluorescence). Signals representing normal and compromomised cells are labeled.

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. 

Assays that measure mitochondrial function and cell viability and growth, cell membrane integrity, membrane potential, intracellular ATP, reduced glutathione... 

As mentioned above, cytotoxicity evaluation is one of the applications on iPSC-derived disease models [104]. With the use of a high-content assay, a series of readouts, such as cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis, have been examined as a general and mechanism-specific hepatotoxicity. In this particular application, the cell model for the assay... 

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

Farrelly, E. Amaral, M. C. Marshall, L. Huang, S. G. A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells. Anal Biochem. 2001, 293, 269-276. 

Jacohson, J. Duchen, M. R. Heales, S. J. R. Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential imphcations for assays of cardioliponyl acridine orangein and mitochondrial mass. J. Neurochem. 2002, 82, 224—233. 

Staining Applications Mitochondria cells Biological Applications Detecting mitochondrial membrane potential apoptosis assays multi-drug resistance assays ... 

Jensen, K. H. R. Rekling, J. C. Development of a no-wash assay for mitochondrial membrane potential using the styryl dye DASPEI. J. Biomol. Screen. 2010,15,1071-1081. 

Huang, S. Chen, J. High-throughput screening assays for modulators of mitochondrial membrane potential. PCT Int. Appl. WO 2000068686, 2000. 

Silver NPs (15 and lOOnm) were evaluated for their potential toxicity in vitro in BRL 3A rat liver cells [10], In order to evaluate such toxicity, the cellular morphology, mitochondrial function MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, membrane leakage of lactate dehydrogenase (LDH), reduced glutathione (GSH) levels, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were assessed during a 24-h exposure period. 

MTT assay is a standard colorimetric assay used to determine cytotoxicity of potential medicinal agents and other toxic materials. It is based on the reduction of the tetrazolium salt MTT by viable cells. A mitochondrial dehydrogenase enzyme is able to cleave the tetrazolium rings of the pale yellow MTT and form dark purple formazan crystals, which are largely impermeable to cell membranes resulting in the accumulation of these crystals within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of crystals, which are solubilized. The metabolic activity of cells is directly proportional to the concentration of the created formazan product (22), whose color is quantified in a colorimetric assay. 

---