title: Application of a homogenous membrane potential assay to assess mitochondrial function.
abstract: 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.
authors.affiliation: NIH Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.
authors.forename: Srilatha
authors.initials: S
authors.lastname: Sakamuru
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.forename: Matias S
authors.initials: MS
authors.lastname: Attene-Ramos
authors.forename: Ruili
authors.initials: R
authors.lastname: Huang
authors.forename: Jianming
authors.initials: J
authors.lastname: Lu
authors.forename: Louie
authors.initials: L
authors.lastname: Shou
authors.forename: Min
authors.initials: M
authors.lastname: Shen
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tice
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Menghang
authors.initials: M
authors.lastname: Xia
mesh_terms: D001562:Benzimidazoles; D002232:Carbocyanines; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004867:Equipment Design; D005456:Fluorescent Dyes; D005470:Fluorometry; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D049691:History, Medieval; D006801:Humans; D020128:Inhibitory Concentration 50; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D008904:Miniaturization; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015394:Molecular Structure; D015203:Reproducibility of Results; D020112:Rhodamine 123; D012235:Rhodamines; D013329:Structure-Activity Relationship
chemical_list: C558346:1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloro-benzimidazolocarbocyanine iodide; D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123; C005358:tetramethylrhodamine