Seahorse Xfe24 Extracellular Flux Analyzer‐Based Analysis of Cellular Respiration in Caenorhabditis elegans

Anthony L. Luz1, Latasha L. Smith2, John P. Rooney1, Joel N. Meyer1

1 Duke University, Durham, North Carolina, 2 Duke University School of Medicine, Durham, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.7
DOI:  10.1002/0471140856.tx2507s66
Online Posting Date:  November, 2015
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Abstract

Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and intercellular as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler), and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters [basal oxygen consumption rate (OCR), ATP‐linked respiration, maximal OCR, spare respiratory capacity, and proton leak] of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. © 2015 by John Wiley & Sons, Inc.

Keywords: mitochondrial toxicity; mitochondrial respiration; Seahorse XFe24; Caenorhabditis elegans

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vivo Quantification of the Fundamental Parameters of the Mitochondrial Electron Transport Chain in Larval Stage Four Nematodes
  • Support Protocol 1: Age‐Synchronizing Nematodes via Sodium Hypochlorite Treatment
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In Vivo Quantification of the Fundamental Parameters of the Mitochondrial Electron Transport Chain in Larval Stage Four Nematodes

  Materials
  • XF Flux pack including:
  • XFe24 extracellular flux assay kits (each kit contains a sensor cartridge, lid, hydrobooster, and 24‐well utility plate)
  • XF Calibrant
  • XF cell culture microplates
  • OP50 K agar plates with synchronous populations of L4 nematodes (see Support Protocol)
  • 80 mM sodium azide dissolved in unbuffered EPA water (see recipe for unbuffered EPA water; store at 4°C)
  • 1.25 mM carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP; Sigma‐Aldrich, cat. no. C2920; dissolved in DMSO, store at −20°C)
  • 2.0 mM dicyclohexylcarbodiimide (DCCD; Sigma‐Aldrich, cat. no. 36650; dissolved in DMSO, store at −20°C)
  • K medium (see recipe)
  • 0.1% (v/v) Triton X‐100 (diluted in distilled deionized H 2O; store at room temperature)
  • Unbuffered EPA water (see recipe)
  • 10% sodium dodecyl sulfate (SDS) solution (dissolved in distilled deionized H 2O; store at room temperature for up to 6 months)
  • Thermo Scientific Pierce BCA Protein Assay Kit
  • Seahorse XFe24 Extracellular Flux Analyzer and computer (provided with flux analyzer with XFe24 Wave software installed)
  • 15‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Centrifuge
  • Orbital shaker
  • 20°C incubator
  • Glass microscope slides
  • Dissecting light microscope
  • Ultrasonicator (Ultrasonic homogenizer, Model 3000, Biologics, Inc.)
  • Microplate reader (FLUOstar OPTIMA, BMG Labtech), with filters capable of measuring absorbance in the range of 540 to 590 nm

Support Protocol 1: Age‐Synchronizing Nematodes via Sodium Hypochlorite Treatment

  Materials
  • OP50‐seeded K agar plates containing gravid adult nematodes: C. elegans strains available through the Caenorhabditis elegans Genetics Center (CGC, University of Minnesota); instructions for the preparation of K agar plates detailed in Lewis and Fleming ( )
  • K medium (see recipe)
  • 70% ethanol
  • Sodium hydroxide bleach solution (see recipe)
  • Complete K medium (see recipe)
  • 15‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Dissecting light microscope
  • L‐shaped glass rod
  • Centrifuge
  • Bunsen burner
  • Orbital shaker
  • 20°C incubator
  • 50‐ml cell culture flask
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Figures

Videos

Literature Cited

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