Analysis of Mitochondrial Dysfunction During Cell Death

Vladimir Gogvadze1, Sten Orrenius1, Boris Zhivotovsky1

1 Institute of Environmental Medicine, Karolinska Institutet, Stockholm
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.10
DOI:  10.1002/0471140856.tx0210s19
Online Posting Date:  May, 2004
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Abstract

Attempts to identify a common underlying step in the apoptotic program in response to various cytotoxic stimuli have focused on the role of mitochondria in this form of cell death. This unit contains a family of protocols that can be used to assess mitochondrial functions during apoptotic responses. Protocols are included for the collection and analysis of released proteins, for detection of the mitochondrial permeability transition, for measurement of mitochondrial membrane potential, and for preparation of mitochondria from different tissue sources.

     
 
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Table of Contents

  • Evaluation of the Release of Proteins from Intermembrane Space of Mitochondria
  • Basic Protocol 1: Collecting Samples of Proteins from Isolated Mitochondria
  • Basic Protocol 2: Evaluation of Cytochrome c Release from the Mitochondria of Apoptotic Cells
  • Basic Protocol 3: Immunoblot Analysis of Proteins Released from the Mitochondria During Apoptosis
  • Basic Protocol 4: Assessment of the Mitochondrial Membrane Potential in Apoptotic Cells
  • Basic Protocol 5: Microscopy of Apoptotic Cells Stained with MitoTracker
  • Assessment of the Mitochondrial Permeability Transition in Isolated Mitochondria
  • Basic Protocol 6: Monitoring of Ca2+ Fluxes Across the Inner Mitochondrial Membrane with a Ca2+‐Sensitive Electrode
  • Alternate Protocol 1: Monitoring of Ca2+ Fluxes Across the Inner Mitochondrial Membrane with a Spectrophotometer
  • Determination of Mitochondrial Membrane Potential
  • Basic Protocol 7: Measurements of Mitochondrial Membrane Potential with a TPP+‐Sensitive Electrode
  • Alternate Protocol 2: Measurement of Mitochondrial Membrane Potential Using a Spectrophotometer
  • Basic Protocol 8: Estimation of Mitochondrial Swelling
  • Basic Protocol 9: Estimation of Mitochondrial Ca2+ Accumulation in Digitonin‐Permeabilized Cells
  • Isolation of Mitochondria
  • Support Protocol 1: Isolation of Rat Liver Mitochondria
  • Support Protocol 2: Isolation of Brain Mitochondria
  • Support Protocol 3: Isolation of Mitochondria from Cultured Cells
  • Support Protocol 4: Estimation of the Quality of Isolated Mitochondria: Measuring the Respiratory Control Ratio (RCR)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Collecting Samples of Proteins from Isolated Mitochondria

  Materials
  • Isolated mitochondria (Support Protocols protocol 121 to protocol 143)
  • Buffer

Basic Protocol 2: Evaluation of Cytochrome c Release from the Mitochondria of Apoptotic Cells

  Materials
  • Cells of interest (e.g., Jurkat cells, U 937, HeLa)
  • Apoptotic stimuli (e.g., etoposide, staurosporine)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • S100 buffer (see recipe), ice‐cold
  • Refrigerated low‐speed centrifuge and ultracentrifuge
  • Additional reagents and equipment for immunoblot analysis (see protocol 3 and Gallagher et al., )

Basic Protocol 3: Immunoblot Analysis of Proteins Released from the Mitochondria During Apoptosis

  Materials
  • Sample collected from apoptotic mitochondria (see protocol 1 or protocol 22)
  • 4× Laemmli's loading buffer (see recipe)
  • 15% (w/v) SDS‐PAGE gel ( appendix 3F)
  • 5% (w/v) nonfat milk in PBS (see appendix 2A for PBS)
  • Antibody specific for apoptotic protein of interest (e.g., Becton Dickinson Biosciences)
  • PBS ( appendix 2A) containing 2.5% (w/v) nonfat dry milk
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 1% (w/v) bovine serum albumin and 0.01% (w/v) azide (NaN 3)
  • PBS ( appendix 2A) containing 15% (v/v) Tween 20
  • Horseradish peroxidase–conjugated secondary antibody (e.g., Pierce)
  • ECL Western Blotting Detection Reagents kit (Amersham Biosciences)
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3F) and electroblotting (Gallagher et al., )

Basic Protocol 4: Assessment of the Mitochondrial Membrane Potential in Apoptotic Cells

  Materials
  • Cells of interest (e.g., Jurkat cells, U 937, HeLa)
  • RPMI‐1640 medium (Life Technologies) supplemented with 5% (v/v) heat‐inactivated fetal bovine serum, 2 mM L‐glutamine, penicillin (100 U/ml), and streptomycin (100 µg/ml)
  • 25 mM TMRE stock solution: dissolve 12.8 mg tetramethylrhodamine methyl ester (TMRE; Molecular Probes) in 1 ml ethanol; store per manufacturer's instructions
  • HEPES buffer (see recipe)
  • Flow cytometer (e.g., FACS; Becton Dickinson)

Basic Protocol 5: Microscopy of Apoptotic Cells Stained with MitoTracker

  Materials
  • Cells of interest (e.g., Jurkat cells, U 937, HeLa) and appropriate culture medium
  • MitoTracker Red (Molecular Probes); store according the manufacturer's instructions
  • Dimethylsulfoxide (DMSO)
  • 4% (w/v) paraformaldehyde in serum‐free culture medium
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • PBS ( appendix 2A) containing 0.2% (v/v) Triton X‐100
  • PBS ( appendix 2A) containing 1% (v/v) fetal bovine serum (FBS)
  • Mounting medium: 50% (w/v) glycerol/PBS
  • Fluorescent or scanning confocal microscope (e.g., Bio‐Rad), with 581‐nm excitation filter and 644‐nm emission filter
  • Additional reagents and equipment for cell culture ( appendix 3B)

Basic Protocol 6: Monitoring of Ca2+ Fluxes Across the Inner Mitochondrial Membrane with a Ca2+‐Sensitive Electrode

  Materials
  • Incubation buffer for calcium‐sensitive electrode (see recipe)
  • Isolated mitochondria ( protocol 12, protocol 132, or protocol 143)
  • 2.5 mM rotenone: dissolve 1 mg rotenone (Sigma) in 1 ml ethanol; store frozen at −20°C up to 1 month
  • 10 mM CaCl 2: dissolve 1.47 mg CaCl 2·2H 2O in 1 ml H 2O; store frozen at −20°C up to 1 month
  • 0.5 M KH 2PO 4: dissolve 136.1 mg KH 2PO 4 in 2 ml H 2O and adjust pH to 7.4 with KOH; store frozen at −20°C up to 1 month
  • Apparatus for membrane potential measurement consisting of:
    • Glass (or plastic) sample chamber large enough for incubation volume, which can be warmed by being connected to a water bath and can be constantly stirred
    • Ca2+‐sensitive electrode (e.g., Orion)
    • pH meter as source of current
    • Chart recorder with variable input and chart speed
    • Any commercially available reference electrode for pH measurements

Alternate Protocol 1: Monitoring of Ca2+ Fluxes Across the Inner Mitochondrial Membrane with a Spectrophotometer

  • 25 mM arsenazo III: dissolve 19.4 mg arsenazo III (Sigma) in 1 ml H 2O; store frozen at −20°C up to 1 month
  • Dual‐wavelength recording spectrophotometer set at 675 versus 685 nm, with appropriate cuvettes

Basic Protocol 7: Measurements of Mitochondrial Membrane Potential with a TPP+‐Sensitive Electrode

  Materials
  • Incubation buffer for TPP+‐sensitive electrode (see recipe)
  • TPP+ stock solution: dissolve 3.75 mg tetraphenylphosphonium chloride (Aldrich) in 10 ml H 2O; store up to 1 month at room temperature
  • Isolated mitochondria ( protocol 12, protocol 132, or protocol 143)
  • 2.5 mM rotenone: dissolve 1 mg rotenone (Sigma) in 1 ml ethanol; store frozen at −20°C up to 1 month
  • 10 mM CaCl 2: dissolve 1.47 mg CaCl 2·2H 2O in 1 ml H 2O; store frozen at −20°C up to 1 month
  • 0.5 M KH 2PO 4: dissolve 136.1 mg KH 2PO 4 in 2 ml H 2O and adjust pH to 7.4 with KOH; store frozen at −20°C up to 1 month
  • Apparatus for membrane potential measurement consisting of:
    • Glass (or plastic) sample chamber that can accommodate 2 ml of incubation buffer, which can be warmed by being connected to a water bath and can be constantly stirred
    • TPP+ electrode (purchase from Microelectrodes, Inc. or prepare in the laboratory, Kamo et al., )
    • pH meter as source of current
    • Chart recorder with variable input and chart speed
    • Any commercially available reference electrode

Alternate Protocol 2: Measurement of Mitochondrial Membrane Potential Using a Spectrophotometer

  • Incubation buffer for calcium‐sensitive electrode (see recipe)
  • 10 mM safranin: dissolve 3.5 mg safranin in 1 ml H 2O; store frozen at −20°C up to 1 month
  • Dual‐wavelength recording spectrophotometer set at 511 versus 533 nm, with appropriate cuvettes

Basic Protocol 8: Estimation of Mitochondrial Swelling

  Materials
  • Incubation buffer for swelling (see recipe)
  • Isolated mitochondria ( protocol 12, protocol 132, or protocol 143)
  • 10 mM CaCl 2: dissolve 1.47 mg CaCl 2·2H 2O in 1 ml H 2O; store frozen at −20°C up to 1 month
  • 2.5 mM rotenone: dissolve 1 mg rotenone (Sigma) in 1 ml ethanol; store frozen at −20°C up to 1 month
  • 0.5 M KH 2PO 4: dissolve 136.1 mg KH 2PO 4 in 2 ml H 2O and adjust pH to 7.4 with KOH; store frozen at −20°C up to 1 month
  • Spectrophotometer with 540‐nm filter, or multiwavelength spectrophotometer
  • Chart recorder with variable input and chart speed

Basic Protocol 9: Estimation of Mitochondrial Ca2+ Accumulation in Digitonin‐Permeabilized Cells

  Materials
  • Cells of interest (Jurkat cells, U 937, HeLa)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Incubation buffer for digitonin‐permeabilized cells (see recipe)
  • 1% (w/v) digitonin: dissolve 10 mg digitonin in 1 ml H 2O; shake vigorously before adding to cells; store up to 2 to 3 months at room temperature
  • 2.5 mM rotenone: dissolve 1 mg rotenone (Sigma) in 1 ml ethanol; store frozen at −20°C up to 1 month
  • 10 mM CaCl 2: dissolve 1.47 mg CaCl 2·2H 2O in 1 ml H 2O; store frozen at −20°C up to 1 month
  • Apparatus for membrane potential measurement consisting of:
    • Glass (or plastic) sample chamber large enough for incubation volume, which can be warmed up by being connected to a water bath and can be constantly stirred
    • Ca2+‐sensitive electrode (e.g., Orion)
    • pH meter as source of current
    • Chart recorder with variable input and chart speed
    • Any commercially available reference electrode for pH measurements

Support Protocol 1: Isolation of Rat Liver Mitochondria

  Materials
  • 150‐ to 200‐g rat
  • Buffer A for liver (see recipe)
  • Buffer B: Buffer A (for liver) without EDTA
  • Dissecting equipment
  • Motor‐driven glass Dounce homogenizer and tight Teflon pestle
  • Refrigerated centrifuge and 50‐ml centrifuge tubes
  • Additional reagents and equipment for protein assay ( appendix 3G) and determination of the respiratory control ratio for mitochondria ( protocol 15)
NOTE: All operations should be done on ice, using ice‐cold buffers and instruments.

Support Protocol 2: Isolation of Brain Mitochondria

  Materials
  • 150‐ to 200‐g rat
  • SET buffer (see recipe), ice‐cold
  • 3% and 6% (w/v) Ficoll solutions (see recipe)
  • MSH buffer (see recipe)
  • Motor‐driven glass Dounce homogenizer and tight Teflon pestle
  • Refrigerated centrifuge and 50‐ml centrifuge tubes
  • Additional reagents and equipment for protein assay ( appendix 3G) and determination of the respiratory control ratio for mitochondria ( protocol 15)

Support Protocol 3: Isolation of Mitochondria from Cultured Cells

  Materials
  • Tissue culture cells (e.g., Jurkat cells, U 937, HeLa; appendix 3B)
  • Complete RPMI medium ( appendix 3B) or appropriate medium for cells
  • Buffer A for cultured cells (see recipe), ice‐cold
  • Buffer B: Buffer A (for cultured cells) without EGTA
  • 1% (w/v) digitonin: dissolve 10 mg digitonin in 1 ml H 2O; shake vigorously before adding to cells; store up to 2 to 3 months at room temperature
  • Refrigerated centrifuge and tubes

Support Protocol 4: Estimation of the Quality of Isolated Mitochondria: Measuring the Respiratory Control Ratio (RCR)

  Materials
  • Mitochondria ( protocol 12, protocol 132, or protocol 143)
  • Incubation buffer for testing mitochondrial quality (see recipe)
  • 2.5 mM rotenone: dissolve 1 mg rotenone (Sigma) in 1 ml ethanol; store frozen at −20°C up to 1 month
  • 0.5 M sodium succinate: dissolve 135 mg of sodium succinate in 1 ml of H 2O, store frozen at −20°C up to 1 month
  • 50 mM ADP: dissolve 23 mg of ADP in 1 ml H 2O and adjust pH to 7.5 with KOH; store frozen at −20°C up to 1 month
  • 1 mM CCCP: dissolve 0.2 mg CCCP in 1 ml ethanol and mix thoroughly; store frozen at −20°C up to 1 month
  • Biological oxygen monitor equipped with a Clark‐type oxygen electrode (Yellow Spring Instrument Co.) or similar equipment (operate per manufacturer's instructions)
  • Chart recorder with variable input and chart speed
  • 10‐ and 50‐µl Hamilton syringes (e.g., Sigma)
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Figures

Videos

Literature Cited

Literature Cited
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   Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Brenner, C., Larochette, N., Prevost, M.C., Alzari, P.M., and Kroemer, G. 1999. Mitochondrial release of caspase‐2 and‐9 during the apoptotic process. J. Exp. Med. 189:381‐394.
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