Measuring Mitochondrial Function in Permeabilized Cells Using the Seahorse XF Analyzer or a Clark‐Type Oxygen Electrode

Ajit S. Divakaruni1, George W. Rogers2, Anne N. Murphy1

1 Department of Pharmacology, University of California, San Diego, La Jolla, California, 2 Seahorse Bioscience, North Billerica, Massachusetts
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.2
DOI:  10.1002/0471140856.tx2502s60
Online Posting Date:  May, 2014
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Abstract

Measurements of mitochondrial respiration in intact cells can help define metabolism and its dysregulation in fields such as cancer, metabolic disease, immunology, and neurodegeneration. Although cells can be offered various substrates in the assay medium, many cell types can oxidize stored pools of energy substrates. A general bioenergetic profile can therefore be obtained using intact cells, but the inability to control substrate provision to the mitochondria can restrict an in‐depth, mechanistic understanding. Mitochondria can be isolated from intact cells, but the yield and quality of the end product is often poor and prone to subselection during isolation. Plasma membrane permeabilization of cells provides a solution to this challenge, allowing experimental control of the medium surrounding the mitochondria. This unit describes techniques to measure respiration in permeabilized adherent cells using a Seahorse XF Analyzer or permeabilized suspended cells in a Hansatech Oxygraph. Curr. Protoc. Toxicol. 60:25.2.1‐25.2.16. © 2014 by John Wiley & Sons, Inc.

Keywords: mitochondria; bioenergetics; perfringolysin O; digitonin; XF PMP

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Respiration in Permeabilized Adherent Cells Using Recombinant Perfringolysin O and the Seahorse XF Analyzer
  • Basic Protocol 2: Measuring Respiration in Permeabilized Cells Using a Clark‐Type Oxygen Electrode
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measuring Respiration in Permeabilized Adherent Cells Using Recombinant Perfringolysin O and the Seahorse XF Analyzer

  Materials
  • Adherent cells of interest
  • XF Calibrant (cat. no. 100840‐000, Seahorse Bioscience)
  • Oxidizable substrates and other reagents (see recipe and Tables 25.2.1 and 25.2.2)
  • 3× MAS buffer (see recipe)
  • Ultrapure or tissue‐culture‐grade H 2O
  • 10% (w/v) fatty acid–free BSA (see recipe) or powdered BSA
  • Recombinant perfringolysin O (rPFO): XF Plasma Membrane Permeabilizer (XF PMP, Seahorse Bioscience, cat. no. 102504‐100)
  • XF24 or XF96 Extracellular Flux Analyzer (Seahorse Bioscience) with 24‐ or 96‐well cell culture microplates and XF Extracellular Flux Assay kit (cartridge and utility plate)
  • 37°C water bath
  • Multichannel pipettors for 20 to 200 µl (XF96) or 50 to 500 µl (XF24); optional but highly recommended
Table 5.2.1   MaterialsOxidizable Substrates and Corresponding Reagents and Inhibitors for Use in Mitochondrial Respiration Assays aPreparation of Stock Solutions for Mitochondrial Respiration Assays

Substrate Final concentration Additives Relevant inhibitors
NADH‐linked (Complex I) substrates
Pyruvate 10 mM b 1.0 mM malate, 2 mM DCA c 2 µM rotenone, 2 µM UK5099
Glutamate 10 mM 10 mM malate d 2 µM rotenone, titrated AOA
β‐Hydroxybutyrate 10 mM 1 mM malate 2 µM rotenone
Palmitoyl carnitine/octanyl carnitine 40 µM 1 mM malate 2 µM antimycin A
Palmitoyl CoA 40 µM 1 mM malate, 0.5 mM carnitine, 1 mM ATP e 40 µM etomoxir, 2 µM antimycin A
Q‐linked (Complex II or III) substrates
Succinate 10 mM 2 µM rotenone 2 µM antimycin A, 2 µM myxothiazol, 20 mM malonate
Glycerol‐3‐phosphate 5 to 10 mM 2 µM rotenone 2 µM antimycin A, 2 µM myxothiazol
Cytochrome oxidase‐linked (Complex IV) substrate
Ascorbate 10 mM 100 µM TMPD, 1 µM antimycin A 20 mM azide
Compound Supplier information Stock concentration Solvent
Oxidizable substrates:
Glutamate a Sigma (G1251) 0.5 to 1 M f H 2O
Malate a Sigma (02288) 0.5 to 1 M f H 2O
Succinate a Sigma (S3674) 0.5 to 1 M f H 2O
Glycerol‐3‐phosphate Sigma (G7886) 0.25 M H 2O
Palmitoyl CoA Sigma (P9716) 10 mM H 2O
Palmitoyl carnitine Sigma (P1645) 10 mM 95% (v/v) ethanol
Octanoyl carnitine Advent Bio (43360) 10 mM 95% (v/v) ethanol
Pyruvate b Sigma (107360) 0.5 to 1 M f H 2O
β‐Hydroxybutyrate b Sigma (166898) 0.5 to 1 M f H 2O
Ascorbate Sigma (5960) 2 M f H 2O
TMPD c Sigma (87890) 10 mM 10 mM ascorbate
Inhibitors and other reagents:
ADP, K+ salt Sigma (A5285) 0.5 M f H 2O
Rotenone Sigma (R8875) d 2 mM 95% (v/v) ethanol
Antimycin A Sigma (A8674) d 2 mM 95% (v/v) ethanol
Oligomycin Sigma (75371) d 5 mM 95% (v/v) ethanol
FCCP e Sigma (C2920) d 10 mM 95% (v/v) ethanol
DCA, K+ salt Sigma (348082) 1 M f H 2O
Carnitine Bachem (F‐2700) 0.5 M f H 2O
ATP Sigma (A26209) 0.5 M f H 2O

 aAbbreviations: AOA, aminooxyacetate; CoA, coenzyme A; DCA, dichloroacetate; TMPD, N,N,N′,N′‐tetramethyl‐p‐phenylenediamine.
 bTo look at activity of the mitochondrial pyruvate carrier, use ≤5 mM pyruvate to minimize effects of non‐facilitated pyruvate uptake.
 cDCA is used to relieve potential kinase inhibition of the pyruvate dehydrogenase (PDH) complex. Omit if interested in conditions where PDH can control respiration rate.
 dAn equimolar concentration of malate is added to ensure maximal aminotransferase activity.
 eATP is included to minimize potential loss of palmityol CoA by de‐esterification. An uncoupler‐stimulated rate of respiration can be obtained under these conditions. ATP can be omitted if a state 3 rate is desired.
Table 5.2.2   MaterialsOxidizable Substrates and Corresponding Reagents and Inhibitors for Use in Mitochondrial Respiration Assays aPreparation of Stock Solutions for Mitochondrial Respiration Assays

Substrate Final concentration Additives Relevant inhibitors
NADH‐linked (Complex I) substrates
Pyruvate 10 mM b 1.0 mM malate, 2 mM DCA c 2 µM rotenone, 2 µM UK5099
Glutamate 10 mM 10 mM malate d 2 µM rotenone, titrated AOA
β‐Hydroxybutyrate 10 mM 1 mM malate 2 µM rotenone
Palmitoyl carnitine/octanyl carnitine 40 µM 1 mM malate 2 µM antimycin A
Palmitoyl CoA 40 µM 1 mM malate, 0.5 mM carnitine, 1 mM ATP e 40 µM etomoxir, 2 µM antimycin A
Q‐linked (Complex II or III) substrates
Succinate 10 mM 2 µM rotenone 2 µM antimycin A, 2 µM myxothiazol, 20 mM malonate
Glycerol‐3‐phosphate 5 to 10 mM 2 µM rotenone 2 µM antimycin A, 2 µM myxothiazol
Cytochrome oxidase‐linked (Complex IV) substrate
Ascorbate 10 mM 100 µM TMPD, 1 µM antimycin A 20 mM azide
Compound Supplier information Stock concentration Solvent
Oxidizable substrates:
Glutamate a Sigma (G1251) 0.5 to 1 M f H 2O
Malate a Sigma (02288) 0.5 to 1 M f H 2O
Succinate a Sigma (S3674) 0.5 to 1 M f H 2O
Glycerol‐3‐phosphate Sigma (G7886) 0.25 M H 2O
Palmitoyl CoA Sigma (P9716) 10 mM H 2O
Palmitoyl carnitine Sigma (P1645) 10 mM 95% (v/v) ethanol
Octanoyl carnitine Advent Bio (43360) 10 mM 95% (v/v) ethanol
Pyruvate b Sigma (107360) 0.5 to 1 M f H 2O
β‐Hydroxybutyrate b Sigma (166898) 0.5 to 1 M f H 2O
Ascorbate Sigma (5960) 2 M f H 2O
TMPD c Sigma (87890) 10 mM 10 mM ascorbate
Inhibitors and other reagents:
ADP, K+ salt Sigma (A5285) 0.5 M f H 2O
Rotenone Sigma (R8875) d 2 mM 95% (v/v) ethanol
Antimycin A Sigma (A8674) d 2 mM 95% (v/v) ethanol
Oligomycin Sigma (75371) d 5 mM 95% (v/v) ethanol
FCCP e Sigma (C2920) d 10 mM 95% (v/v) ethanol
DCA, K+ salt Sigma (348082) 1 M f H 2O
Carnitine Bachem (F‐2700) 0.5 M f H 2O
ATP Sigma (A26209) 0.5 M f H 2O

 aThe corresponding acid is purchased as a powder.
 bThe corresponding acid is purchased as a liquid. Concentrations of powdered sodium salts at ≥10 mM are avoided to prevent mitochondrial calcium efflux via the Na+/Ca2+ exchanger).
 cN,N,N′,N′‐Tetramethyl‐p‐phenylenediamine (TMPD) is stored with 10 mM ascorbate to ensure its reduction.
 dAlso available from Seahorse Bioscience and other vendors.
 eAbbreviations: DCA, dichloroacetate; FCCP, carbonyl cyanide 4‐(trifluoromethoxy)phenyl‐hydrazone.
 fAdjust pH to 7.2 with KOH.

Basic Protocol 2: Measuring Respiration in Permeabilized Cells Using a Clark‐Type Oxygen Electrode

  Materials
  • Air‐saturated, deionized water, 37°C
  • Sodium dithionite (sodium hydrosulfite, Sigma 157953)
  • Cell suspension and growth medium
  • KCl buffer (optional; see recipe)
  • 10% (w/v) digitonin (optional; see recipe)
  • Tissue‐culture‐grade or deionized water
  • 95% (v/v) ethanol
  • Hansatech Oxytherm System, including Oxytherm electrode control unit, S1/Minielectrode disc, personal computer with Oxygraph Plus data acquisition software, chamber stir bar, half‐saturated KCl solution, filter paper, membrane applicator, O‐rings, and PTFE membrane
  • Liquid aspiration apparatus
  • Microsyringes (e.g., Hamilton) or other narrow‐tip pipets to transfer small volumes (0.01 to 25 µl) through the chamber stopper
  • Additional reagents and equipment described for use with the Seahorse XF Analyzer (see protocol 1)
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Figures

Videos

Literature Cited

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Internet Resources
  http://www.seahorsebio.com/learning/webinars/details.php?wID=50
  A webinar given by the authors, in conjunction with Seahorse Bioscience, including background information on permeabilized cells and a tutorial on identifying mitochondrial mechanisms of drug action using permeabilized cells in the XF Analyzer.
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