| Application of a homogenous membrane potential assay to assess mitochondrial function. | |
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MedLine Citation:
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PMID: 22433785 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Decreases in mitochondrial membrane potential (MMP) have been associated with mitochondrial dysfunction that could lead to cell death. The MMP is generated by an electrochemical gradient via the mitochondrial electron transport chain coupled to a series of redox reactions. Measuring the MMP in living cells is commonly used to assess the effect of chemicals on mitochondrial function; decreases in MMP can be detected using lipophilic cationic fluorescent dyes. To identify an optimal dye for use in a high-throughput screening (HTS) format, we compared the ability of mitochondrial membrane potential sensor (Mito-MPS), 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide, rhodamine 123, and tetramethylrhodamine to quantify a decrease in MMP in chemically exposed HepG2 cells cultured in 1,536-well plates. Under the conditions used, the optimal dye for this purpose is Mito-MPS. Next, we developed and optimized a homogenous cell-based Mito-MPS assay for use in 1,536-well plate format and demonstrated the utility of this assay by screening 1,280 compounds in the library of pharmacologically active compounds in HepG2 cells using a quantitative high-throughput screening platform. From the screening, we identified 14 compounds that disrupted the MMP, with half-maximal potencies ranging from 0.15 to 18 μM; among these, compound clusters that contained tyrphostin and 3'-substituted indolone analogs exhibited a structure-activity relationship. Our results demonstrate that this homogenous cell-based Mito-MPS assay can be used to evaluate the ability of large numbers of chemicals to decrease mitochondrial function. |
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Authors:
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Srilatha Sakamuru; Xiao Li; Matias S Attene-Ramos; Ruili Huang; Jianming Lu; Louie Shou; Min Shen; Raymond R Tice; Christopher P Austin; Menghang Xia |
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Publication Detail:
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Type: Comparative Study; Evaluation Studies; Historical Article; Journal Article; Research Support, N.I.H., Intramural Date: 2012-03-20 |
Journal Detail:
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Title: Physiological genomics Volume: 44 ISSN: 1531-2267 ISO Abbreviation: Physiol. Genomics Publication Date: 2012 May |
Date Detail:
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Created Date: 2012-05-03 Completed Date: 2012-08-31 Revised Date: 2013-05-02 |
Medline Journal Info:
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Nlm Unique ID: 9815683 Medline TA: Physiol Genomics Country: United States |
Other Details:
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Languages: eng Pagination: 495-503 Citation Subset: IM |
Affiliation:
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NIH Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland 20892-3370, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Benzimidazoles* Carbocyanines* Cell Survival / drug effects Dose-Response Relationship, Drug Equipment Design Fluorescent Dyes* Fluorometry Hep G2 Cells High-Throughput Screening Assays / instrumentation, methods* History, Medieval Humans Inhibitory Concentration 50 Membrane Potential, Mitochondrial / drug effects* Microscopy, Fluorescence Miniaturization Mitochondria / drug effects*, metabolism, pathology Molecular Structure Reproducibility of Results Rhodamine 123 Rhodamines Structure-Activity Relationship |
| Grant Support | |
ID/Acronym/Agency:
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Y3-ES-7020-01/ES/NIEHS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloro-benzimidazolocarbocyanine iodide; 0/Benzimidazoles; 0/Carbocyanines; 0/Fluorescent Dyes; 0/Rhodamines; 62669-70-9/Rhodamine 123; 62669-72-1/tetramethylrhodamine |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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