High‐Resolution Respirometry for Mitochondrial Characterization of Ex Vivo Mouse Tissues

Carles Cantó1, Pablo M. Garcia‐Roves2

1 Nestlé Institute of Health Sciences S.A, Lausanne, 2 Diabetes and Obesity Research Laboratory, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS) and Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Barcelona
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140061
Online Posting Date:  June, 2015
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Abstract

This article describes methodologies to examine mitochondrial respiration in fresh preparations of mouse tissues, including skeletal muscle, heart, liver, white and brown adipose tissue, and brain. Reference values and tips to maximize experimental efficiencies are also provided. Finally, correction methods and complementary techniques to properly interpret the results are presented and contrasted. © 2015 by John Wiley & Sons, Inc.

Keywords: mitochondrial physiology; bioenergetics; respiratory complexes

     
 
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Table of Contents

  • Introduction
  • Tissue Extraction, Storage, and Preparation
  • Basic Protocol 1: Skeletal Muscle Preparation for Respirometry Analysis
  • Basic Protocol 2: Preparation of Heart Tissue for Respirometry Studies
  • Alternate Protocol 1: Homogenization of Cardiac Tissue Using the PBI Shredder
  • Basic Protocol 3: Preparation of White Adipose Tissue for Respirometry Studies
  • Basic Protocol 4: Preparation of Brown Adipose Tissue for Respirometry Studies
  • Basic Protocol 5: Preparation of Liver Tissue for Respirometry Studies
  • Basic Protocol 6: Preparation of Brain Tissue for Respirometry Studies
  • Basic Protocol 7: Respirometry Assay
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Skeletal Muscle Preparation for Respirometry Analysis

  Materials
  • C57BL/6 J male mice, 12 weeks old, between 25 and 30 g body weight (Charles River Laboratories)
  • Isoflurane (inhalant anesthetic) or injectable solution, e.g., Rodent Combo Anesthetic III (ketamine 37.6 mg/ml; xylazine 1.92 mg/ml; acepromazine 0.38 mg/ml)
  • BIOPS (see recipe in Table 14.0.6100), ice cold
  • Saponin (Sigma, cat. no. S7900)
  • Mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100)
  • 95% O 2, 5% CO 2 medical gas mixture
  • Plastic 12‐well plate (Corning, cat. no. CLS3512)
  • Surgical instruments:
    • Scalpel handle [Fine Science Tools (FST), cat. no. 91003‐12]
    • Needle holder (FST, cat. no. 12503‐15)
    • Small scissors [Marcel Blanc et Cie SA (MB), cat. no. 3250.11]
    • Medium scissors (FST, cat. no. 91401‐12)
    • Big scissors (MB, cat. no. 3010.14)
    • Forceps (MB, cat. no. 2000.11)
    • Small forceps (FST, cat. no. 91106‐12)
    • Two thin‐edged forceps (MB, cat. no. 2240.05)
  • Filter paper
  • Analytical balance (Mettler‐Toledo Intl., cat. no. XA 105DU)
  • O2k Oxygraph high‐resolution respirometer (Oroboros Inc.)

Basic Protocol 2: Preparation of Heart Tissue for Respirometry Studies

  Materials
  • C57BL/6 J male mice, 12 weeks old, between 25 and 30 g body weight (Charles River Laboratories)
  • Isoflurane (inhalant anesthetic) or injectable solution, e.g., Rodent Combo Anesthetic III (ketamine 37.6 mg/ml; xylazine 1.92 mg/ml; acepromazine 0.38 mg/ml)
  • BIOPS (see recipe in Table 14.0.6100), ice cold
  • Saponin (Sigma, cat. no. S7900)
  • Mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100)
  • Magnifying lens
  • Plastic 12‐well plate (Corning, cat. no. CLS3512)
  • Surgical instruments:
    • Scalpel handle [Fine Science Tools (FST), cat. no. 91003‐12]
    • Needle holder (FST, cat. no. 12503‐15)
    • Small scissors [Marcel Blanc et Cie SA (MB), cat. no. 3250.11]
    • Medium scissors (FST, cat. no. 91401‐12)
    • Big scissors (MB, cat. no. 3010.14)
    • Forceps (MB, cat. no. 2000.11)
    • Small forceps (FST, cat. no. 91106‐12)
    • Two thin‐edged forceps (MB, cat. no. 2240.05)
  • Filter paper
  • Analytical balance (Mettler‐Toledo Intl., cat. no. XA 105DU)
  • 95% O 2, 5% CO 2 medical gas mixture
  • O2k Oxygraph, high‐resolution respirometer (Oroboros Inc.)

Alternate Protocol 1: Homogenization of Cardiac Tissue Using the PBI Shredder

  Materials
  • Mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100)
  • Tissue sample (e.g., myocardium)
  • Shredder‐tubes (FT500 PULSE tubes by Pressure BioSciences, Inc., composed of: modules, part no. 110530‐003; serrated rams, part no. 110532‐004PP; and caps, part no.110531‐004)
  • Filter paper
  • Analytical balance (Mettler‐Toledo Intl., cat. no. XA 105DU)
  • Small scissors [Marcel Blanc et Cie SA (MB) 3250.11]
  • PBI‐Shredder SG3 (Pressure BioSciences)
  • 15‐ or 50‐ml conical centrifuge tubes

Basic Protocol 3: Preparation of White Adipose Tissue for Respirometry Studies

  Materials
  • C57BL/6 J male mice, 12 weeks old, between 25 and 30 g body weight (Charles River Laboratories)
  • Isoflurane (inhalant anesthetic) or injectable solution: e.g., Rodent Combo Anesthetic III (ketamine 37.6 mg/ml; xylazine 1.92 mg/ml; acepromazine 0.38 mg/ml)
  • BIOPS (see recipe in Table 14.0.6100) or mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100), ice cold
  • Dissecting microscope
  • Surgical instruments (see protocol 1)
  • 6‐well plate
  • Filter paper
  • O2k Oxygraph, high‐resolution respirometer (Oroboros Inc.)

Basic Protocol 4: Preparation of Brown Adipose Tissue for Respirometry Studies

  Materials
  • C57BL/6 J male mice, 12 weeks old, between 25 and 30 g body weight (Charles River Laboratories)
  • Isoflurane (inhalant anesthetic) or injectable solution: e.g., Rodent Combo Anesthetic III (ketamine 37.6 mg/ml; xylazine 1.92 mg/ml; acepromazine 0.38 mg/ml)
  • Mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100)
  • Surgical instruments (see protocol 1)
  • Cordless pestle homogenizer (VWR, cat. no. 47747‐370) and micropestle for 1.5‐ and 2‐ml tubes (Eppendorf, cat. no. 022365622)
  • O2k Oxygraph, high‐resolution respirometer (Oroboros Inc.)

Basic Protocol 5: Preparation of Liver Tissue for Respirometry Studies

  Materials
  • C57BL/6 J male mice, 12 weeks old, between 25 and 30 g body weight (Charles River Laboratories)
  • Isoflurane (inhalant anesthetic) or injectable solution: e.g., Rodent Combo Anesthetic III (ketamine 37.6 mg/ml; xylazine 1.92 mg/ml; acepromazine 0.38 mg/ml)
  • Saline: 0.9% (w/v) NaCl
  • BIOPS (see recipe in Table 14.0.6100), ice cold
  • Mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100)
  • Surgical instruments (see protocol 1)
  • Analytical balance (Mettler‐Toledo Intl Inc., cat. no. XA 105DU)
  • Filter paper
  • 12‐well plate
  • Cordless pestle homogenizer (VWR, cat. no. 47747‐370) and micropestle for 1.5‐ and 2‐ml tubes (Eppendorf, cat. no. 022365622)
  • O2k Oxygraph, high‐resolution respirometer (Oroboros Inc.)

Basic Protocol 6: Preparation of Brain Tissue for Respirometry Studies

  Materials
  • C57BL/6 J male mice, 12 weeks old, between 25 and 30 g body weight (Charles River Laboratories)
  • Isoflurane (inhalant anesthetic) or injectable solution: e.g., Rodent Combo Anesthetic III (ketamine 37.6 mg/ml; xylazine 1.92 mg/ml; acepromazine 0.38 mg/ml)
  • BIOPS (see recipe in Table 14.0.6100), ice cold
  • Mitochondrial respiration medium (MIR; see recipe in Table 14.0.6100), ice cold
  • Surgical instruments (see protocol 1)
  • Analytical balance (Mettler‐Toledo Intl Inc., cat. no. XA 105DU)
  • 12‐well plate
  • Cordless pestle homogenizer (VWR, cat. no. 47747‐370) and micropestle for 1.5‐ and 2‐ml tubes (Eppendorf, cat. no. 022365622)
  • O2k Oxygraph, high‐resolution respirometer (Oroboros Inc.)

Basic Protocol 7: Respirometry Assay

  Materials
  • Tissue sample (from Basic Protocols protocol 11 to protocol 76 or protocol 3Alternate Protocol)
  • Chemical stocks as described in Table 14.0.6100:
    • 400 mM L‐malic acid
    • 2 M pyruvic acid, sodium salt
    • 2 M L‐glutamic acid
    • 500 mM ADP
    • 1 M disodium succinate
    • 1 mM FCCP
    • 1 mM rotenone
    • 5 mM antimycin A
    • 5 mg/ml saponin
    • 8.1 mM digitonin
    • 4 mM cytochrome c
  • O2k Oxygraph, high‐resolution respirometer (Oroboros Inc.)
  • Hamilton glass microsyringes: (3 × 10 μl, 3× 25 μl, and 2× 50 μl)
  • Datlab software (http://www.oroboros.at/index.php?datlabsoftware)
  • Statistical analysis software (e.g., Microsoft Office Excel)
Table 4.0.3   MaterialsChemical Stocks for Respirometry Assays

Compound Stock concentration Solvent Storage Comments
L‐Malic acid 400 mM H 2O Store at −20°C Neutralize with 10 N KOH
Pyruvic acid, sodium salt 2 M H 2O Freshly prepared
L‐Glutamic acid 2 M H 2O Store at −20°C Neutralize with 5 N KOH
ADP (adenosine 5’diphosphate, potassium salt) 500 mM H 2O Store at −80°C Neutralize with 5 N KOH. To maintain Mg2+ constant during the respiration measurement, mix ADP with MgCl 2 (0.5 mol/mol ADP).
Succinate disodium salt, hexahydrate 1 M H 2O Store at −20°C Adjust pH to 7 with HCl
FCCP 1 mM Ethanol Store at −20°C
Rotenone 1 mM Ethanol Store at −20°C Difficult to dissolve and light sensitive. Very toxic.
Antimycin A 5 mM Ethanol Store at −20°C Very toxic
Saponin 5 mg/ml BIOPS Freshly prepared
Digitonin 8.1 mM DMSO Store at −20°C Toxic
Cytochrome c 4 mM H 2O Store at −20°C

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Figures

Videos

Literature Cited

Literature Cited
  Andreux, P.A., Houtkooper, R.H., and Auwerx, J. 2013. Pharmacological approaches to restore mitochondrial function. Nat. Rev. Drug Discov. 12:465‐483.
  Cannon, B. and Nedergaard, J. 2009. Thermogenesis challenges the adipostat hypothesis for body‐weight control. Proc. Nutr. Soc. 68:401‐407.
  Gnaiger, E. 2003. Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv. Exp. Med. Biol. 543:39‐55.
  Gnaiger, E. and Kuznetsov, A.V. 2002. Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem. Soc. Trans. 30:252‐258.
  He, J., Watkins, S., and Kelley, D.E. 2001. Skeletal muscle lipid content and oxidative enzyme activity in relation to muscle fiber type in type 2 diabetes and obesity. Diabetes 50:817‐823.
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  Kelley, D.E., He, J., Menshikova, E.V., and Ritov, V.B. 2002. Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes 51:2944‐2950.
  Kraunsoe, R., Boushel, R., Hansen, C.N., Schjerling, P., Qvortrup, K., Stockel, M., Mikines, K.J., and Dela, F. 2010. Mitochondrial respiration in subcutaneous and visceral adipose tissue from patients with morbid obesity. J. Physiol. 588:2023‐2032.
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  Larsen, S., Kraunsoe, R., Gram, M., Gnaiger, E., Helge, J.W., and Dela, F. 2014. The best approach: Homogenization or manual permeabilization of human skeletal muscle fibers for respirometry? Anal. Biochem. 446:64‐68.
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  Simoneau, J.A., Veerkamp, J.H., Turcotte, L.P., and Kelley, D.E. 1999. Markers of capacity to utilize fatty acids in human skeletal muscle: Relation to insulin resistance and obesity and effects of weight loss. FASEB J. 13:2051‐2060.
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