Document Detail

Application of a homogenous membrane potential assay to assess mitochondrial function.
MedLine Citation:
PMID:  22433785     Owner:  NLM     Status:  MEDLINE    
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.
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:
Type:  Comparative Study; Evaluation Studies; Historical Article; Journal Article; Research Support, N.I.H., Intramural     Date:  2012-03-20
Journal Detail:
Title:  Physiological genomics     Volume:  44     ISSN:  1531-2267     ISO Abbreviation:  Physiol. Genomics     Publication Date:  2012 May 
Date Detail:
Created Date:  2012-05-03     Completed Date:  2012-08-31     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  9815683     Medline TA:  Physiol Genomics     Country:  United States    
Other Details:
Languages:  eng     Pagination:  495-503     Citation Subset:  IM    
NIH Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.
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MeSH Terms
Cell Survival / drug effects
Dose-Response Relationship, Drug
Equipment Design
Fluorescent Dyes*
Hep G2 Cells
High-Throughput Screening Assays / instrumentation,  methods*
History, Medieval
Inhibitory Concentration 50
Membrane Potential, Mitochondrial / drug effects*
Microscopy, Fluorescence
Mitochondria / drug effects*,  metabolism,  pathology
Molecular Structure
Reproducibility of Results
Rhodamine 123
Structure-Activity Relationship
Grant Support
Reg. No./Substance:
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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