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Methods, compositions and kits for assaying mitochondrial function

a technology of mitochondrial function and kits, applied in the field of methods, compositions and kits for assaying mitochondrial function, can solve the problems of difficult to establish the concentration of detergents under which mitochondrial function is sustained, and achieve the effect of accurate evaluation of intracellular mitochondrial activities

Inactive Publication Date: 2012-11-29
UNIV OF MASSACHUSETTS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention relates to methods, compositions, and devices for accurately measuring the functions of mitochondria (the energy-producing organelles in cells) without damaging the cellular environment. The methods use a special pore-forming protein called perfringolysin O (PFO) to selectively permeabilize the plasma membrane without disrupting the mitochondrial membranes. This allows for accurate assessments of mitochondrial activities such as uptake and release of substrates and products. The PFO-based methods can be used with different types of cells and buffer conditions, and can also be used to evaluate the effects of exogenous agents on mitochondrial functions. Overall, the invention provides a reliable and reproducible way to measure mitochondrial activities in cells.

Problems solved by technology

Detergents, for example, typically solubilize mitochondrial membranes, and even with careful titrations below <0.01% it is difficult to establish concentrations of detergents under which mitochondrial function is sustained.

Method used

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  • Methods, compositions and kits for assaying mitochondrial function
  • Methods, compositions and kits for assaying mitochondrial function
  • Methods, compositions and kits for assaying mitochondrial function

Examples

Experimental program
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example 1

Experimental Design and Methodology

[0097]PFO-Based Cell Permeabilization Methods for Mitochondrial Function Assays:

[0098]Wild type PFOs and variants have been tested for maximal mitochondrial performance (see Table 1 for examples of PFOs) using methods disclosed herein. Specific assays for different OxPhos components applicable to a wide variety of were developed based on cell permeabilization methods described herein. Both established and primary cells were utilized to test the general applicability of PFO-based assays.

[0099]It has been recognized that a reducing agent may be used to increase shelf life of wild-type PFO. PFO contains only one Cys residue at position 459. To avoid the need of a reducing agent in permeabilization reactions, a Cys free derivative, PFOC459A, has been used. This variant has activity comparable to wild type PFO. A mutant rPFOT319C-V334C provides for conditional cell permeabilization, as it does not form pore in the membrane following insertion until a re...

example 2

Experimental Evaluations Using Different Cell Types

[0107]Similar experimental conditions may be used for assays of Complexes I-V, and OxPhos and ETC / RC capacity and across different cells (see Table 4 for a non-limiting list of cells).

TABLE 4Cells for PFO-based mitochondrial function assays.CategorySubcategoryCellsCell linesAdherent cellsHEK293, C2C12, HepG2, A549, H460, MCF7Adherent cells withINS1, SHSY-5YcoatingNon-adherent cellsCell seeding, spin down, and measurements onsame day with above cellsCells may be grouped with respect to their OxPhos capacity vs. ETC / RCcapacity to provide a reference for each enlisted cell line.Primary cellsAdherent cellsβ-cells, astrocytes,Note: primary cellsneurons, fibroblasts,may require coatinghepatocytes, mammaryof plates (e.g., V7epithelial cells,plates) with PEI ormyocytes / blastssimilar reagents e.g.(cardiac, skeletal),PDL, CellTak.Adipocytes(WAT / BAT), adultstem cellsNon-adherent cellsPeripheral bloodMay involve coatingwith coatingsmonocytesof ...

example 3

Assay Using Digitonin as a Permeabilizing Reagent

[0109]Digitonin concentration and respiration buffers were optimized for maximal ADP-stimulated respiration using succinate as substrate in the presence and absence of Cytochrome c. FIG. 2 shows representative data for conditions for INS1E cells that involve the addition of exogenous Cytochrome c. A Ca2+-free low K+ respiration buffer [20 mM TES pH7.4, 3.5 mM KCL, 120 mM NaCl, 0.4 mM KH2PO4, 1.2 mM Na2SO4, 2 mM MgSO4, 1 mM EGTA with 0.4% fatty acid free BSA] was used unless otherwise specified. Since, the intracellular K+ ion concentration is higher (˜120 mM), Ca2+-free high K+ buffers (25-120 mM NaCl replaced with equimolar KCL in the buffer) were tested to determine if mitochondria performed better. It was found that in certain instances high K+ buffers underperformed compared to the low K+ buffer using INS1E cells. Under the same conditions, the performance of two other commonly used permeabilizing agents, saponin, and alamethicin,...

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Abstract

The invention provides methods, compositions, devices, and kits relating to the use of cholesterol-dependent cytolysins (e.g., PFOs) for measuring intracellular mitochondrial activity.

Description

RELATED APPLICATION[0001]This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. No. 61 / 473,730, filed Apr. 8, 2011, and entitled “Methods, Compositions and Kits for Assaying Mitochondrial Function,” which is incorporated herein by reference in its entirety for all purposes.GOVERNMENT SUPPORT[0002]This work was supported under grant number 5R21NS057224-03, awarded by National Institutes of Health (NIH). The U.S. Government has certain rights in the invention.FIELD OF THE INVENTION[0003]The invention relates to methods for evaluating intracellular functions as well as cell health and viability.BACKGROUND OF THE INVENTION[0004]Mitochondrial dysfunction is at the core of many encephalomyopathies, diabetes and cancer. Inherited mutations in over 100 genes constituting the oxidative phosphorylation (OxPhos) machinery are linked with mitochondrial encephalomyopathies in humans. The diseases resulting from defective OxPhos are also referred to a...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/33C12N15/63C12Q1/02
CPCG01N33/5005C07K14/33G01N33/5079G01N2333/33
Inventor YADAVA, NAGENDRAHEUCK, ALEJANDRO PABLOKIM, CHUL
Owner UNIV OF MASSACHUSETTS
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