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Analysis of Mitochondrial Dysfunction During Cell Death
Publication Name:
Current Protocols in Cell Biology
Unit Number:
Unit 18.5
DOI:
10.1002/0471143030.cb1805s19
Online Posting Date:
August, 2003 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.
Table of Contents
- Unit Introduction
- 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 Ca
2+ Fluxes Across the Inner Mitochondrial Membrane with a Ca2+ -Sensitive Electrode -
Alternate Protocol 1: Monitoring of Ca
2+ 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 Ca
2+ 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
Materials
Basic Protocol 1: Collecting Samples of Proteins from Isolated Mitochondria
Materials
- Isolated mitochondria (
- 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
- S100 buffer (see
- Refrigerated low-speed centrifuge and ultracentrifuge
- Additional reagents and equipment for immunoblot analysis (unit
Basic Protocol 3: Immunoblot Analysis of Proteins Released from the Mitochondria During Apoptosis
Materials
- Sample collected from apoptotic mitochondria (see
- 4× Laemmli's loading buffer (see
- 15% (w/v) SDS-PAGE gel (unit
- 5% (w/v) nonfat milk in PBS (see appendix
- Antibody specific for apoptotic protein of interest (e.g., Becton Dickinson Biosciences)
- PBS (appendix
- Nonfat dry milk
- Phosphate-buffered saline (PBS; appendix
3 ) - PBS (appendix
- Horseradish peroxidase–conjugated secondary antibody (e.g., Pierce)
- ECL Western Blotting Detection Reagents kit (Amersham Biosciences)
- Additional reagents and equipment for SDS-PAGE (unit
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
- 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
- PBS (appendix
- PBS (appendix
- 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 (unit
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
- Isolated mitochondria (
- 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 CaCl2 ·2H2 O in 1 ml H2 O; store frozen at –20°C up to 1 month - 0.5 M KH
2 PO4 : dissolve 136.1 mg KH2 PO4 in 2 ml H2 O 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
- Ca
2+ -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
Additional Materials (also see Basic Protocol 6 )
- 25 mM arsenazo III: dissolve 19.4 mg arsenazo III (Sigma) in 1 ml H
2 O; 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 - TPP
+ stock solution: dissolve 3.75 mg tetraphenylphosphonium chloride (Aldrich) in 10 ml H2 O; store up to 1 month at room temperature - Isolated mitochondria (
- 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 CaCl2 ·2H2 O in 1 ml H2 O; store frozen at –20°C up to 1 month - 0.5 M KH
2 PO4 : dissolve 136.1 mg KH2 PO4 in 2 ml H2 O 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
Additional Materials (also see Basic Protocol 7 )
- Incubation buffer for calcium-sensitive electrode (see
- 10 mM safranin: dissolve 3.5 mg safranin in 1 ml H
2 O; 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
- Isolated mitochondria (
- 10 mM CaCl
2 : dissolve 1.47 mg CaCl2 ·2H2 O in 1 ml H2 O; 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
2 PO4 : dissolve 136.1 mg KH2 PO4 in 2 ml H2 O 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
- Incubation buffer for digitonin-permeabilized cells (see
- 1% (w/v) digitonin: dissolve 10 mg digitonin in 1 ml H
2 O; 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 CaCl2 ·2H2 O in 1 ml H2 O; 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
- Ca
2+ -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
- Buffer B:
- 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
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
- 3% and 6% (w/v) Ficoll solutions (see
- MSH buffer (see
- Motor-driven glass Dounce homogenizer and tight Teflon pestle
- Refrigerated centrifuge and 50-ml centrifuge tubes
- Additional reagents and equipment for protein assay (appendix
Support Protocol 3: Isolation of Mitochondria from Cultured Cells
Materials
- Tissue culture cells (e.g., Jurkat cells, U 937, HeLa; unit
- Complete RPMI medium (unit
- Buffer A for cultured cells (see
- Buffer B: Buffer A (for cultured cells) without EGTA
- 1% (w/v) digitonin: dissolve 10 mg digitonin in 1 ml H
2 O; 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 (
- Incubation buffer for testing mitochondrial quality (see
- 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
2 O, store frozen at –20°C up to 1 month - 50 mM ADP: dissolve 23 mg of ADP in 1 ml H
2 O 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)
Looking for materials? Suppliers Guide
Figures
-
Figure 18.5.1MPT-induced release of cytochrome c from mitochondria incubated in either MSH or KCl buffer. (A) Mitochondria were added to buffer at a concentration of 1 mg/ml. After a 2-min stabilization period, mitochondria were loaded with Ca2+ (50 nmol/mg protein), and MPT was induced by adding 5 mM inorganic phosphate (Pi ). (B) Mitochondrial suspensions from (A) were centrifuged and the resulting supernatants were separated by SDS-PAGE and immunoblotted as described in -
Figure 18.5.2Ca2+ -induced release of cytochrome c from mitochondria in the absence of observable MPT. (A) Mitochondria (1 mg/ml) were incubated in KCl-based buffer. After a 2-min stabilization period, mitochondria were loaded with Ca2+ (25 nmol/mg protein) prior to the addition of 1 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 5 min. (B) The amount of cytochrome c released from mitochondria after 1 and 5 min of Ca2+ retention in the presence and absence of 0.5 mM ADP or 1 mM Mg2+ . (C) Samples incubated under the same conditions as (A) were used to evaluate the rate of uncoupled respiration after 1 and 5 min. (D) Samples incubated under the same conditions as conditions as (B) were used to determine the effect of inhibitors of MPT on the rate of uncoupled respiration of Ca2+ -loaded mitochondria. -
Figure 18.5.3The effect of Ca2+ loading on mitochondrial swelling and the release of cytochrome c. Mitochondria (0.5 mg/ml) were incubated in 2 ml of KCl-based buffer. (A) Mitochondria were loaded sequentially with varied amounts of Ca2+ until MPT was induced. (B) Samples were taken after 5 min (lanes 1 to 6) or 8 min (lane 7) of incubation and the supernatants evaluated for cytochrome c content. -
Figure 18.5.4The release of cytochrome c from isolated rat liver mitochondria as a result of mitochondrial permeability transition (MPT). Mitochondria (1 mg/ml) were incubated in KCl-based buffer. After a 2-min stabilization period, mitochondria were loaded with Ca2+ , and, when accumulation was complete, MPT was induced by 5 mM KH2 PO4 . A 200-µl aliquot of mitochondrial suspension was centrifuged and the resulting supernatant and pellet were separated by SDS-PAGE and immunoblotted as described under in2+ loading. -
Figure 18.5.5A typical image of FACS analysis of mitochondrial membrane potential in the cells undergoing apoptosis. Apoptosis was induced in Jurkat cells by 1 µM staurosporine and samples were analyzed 3 hr after initiation of apoptosis. -
Figure 18.5.6MPT monitored with a Ca2+ -sensitive electrode. Mitochondria (1 mg/ml) were incubated in KCl-based buffer. After a 1-min stabilization period, mitochondria were loaded with Ca2+ , and, when accumulation was complete, MPT was induced by 5 mM KH2 PO4 . -
Figure 18.5.7MPT monitored with a -sensitive electrode. Mitochondria (1 mg/ml) were incubated in KCl-based buffer. After a 1-min stabilization period mitochondria were loaded with Ca2+ , and, when accumulation was complete and potential was restored, MPT was induced by 5 mM KH2 PO4 . The concentration of TPP+ was 2 µM. -
Figure 18.5.8MPT assessed by mitochondrial swelling. Mitochondria (0.5 mg/ml) were incubated in 2 ml of KCl-based buffer. Mitochondria were loaded with Ca2+ and 5 mM phosphate was added. Swelling was monitored by a decrease of optical density at 540 nm. -
Figure 18.5.9Estimation of mitochondrial Ca2+ accumulation in digitonin-permeabilized cells. Jurkat cells (2.5 × 106 ) were washed in PBS, resuspended in 500 µl of KCl-based buffer, and added to the incubation chamber. Following a 2-min stabilization period, cells were permeabilized with 0.005% digitonin, and 5 µM rotenone was added in order to maintain pyridine nucleotides in a reduced form. MPT was induced by sequential additions of Ca2+ and changes in the level of this cation were monitored using a Ca2+ -selective electrode. -
Figure 18.5.10Estimation of mitochondrial respiration as described in
Literature Cited
| Literature Cited | |
| Bossy-Wetzel, E., Newmeyer, D.D., and Green, D.R. 1998. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17:37-49. | |
| Crompton, M. 1999. The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341:233-249. | |
| Du, C., Fang, M., Li, Y., Li, L., and Wang, X. 2000. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by elimination IAP inhibition. Cell 102:33-42. | |
| Eskes, R., Desagher, S., Antonsson, B., and Martinou, J.C. 2000. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell Biol. 20:929-935. | |
| Gogvadze, V., Robertson, J.D., Zhibotovsky, B., and Orrenius, S. 2001. Cytochrome c release occurs via Ca | |
| Green, D.R. and Reed, J.C. 1998. Mitochondria and apoptosis. Science 281:1309-1312. | |
| Kamo, N., Muratsugu, M., Hongoh, R., and Kobatake, Y. 1979. Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J. Membr. Biol. 49:105-121. | |
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| Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S., and Wang, X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479-489. | |
| Mancini, M., Nicholson, D.W., Roy, S., Thornberry, N.A., Peterson, E.P., Casciola-Rosen, L.A., and Rosen, A. 1998. The caspase-3 precursor has a cytosolic and mitochondrial distribution: Implications for apoptotic signaling. J. Cell Biol. 140:1485-1495. | |
| Mitchell, P. and Moyle, J. 1967. Chemiosmotic hypothesis of oxidative phosphorylation. Nature 213:137-139. | |
| Petit, P.X., Zamzami, N., Vayssiere, J.L., Mignotte, B., Kroemer, G., and Castedo, M. 1997. Implication of mitochondria in apoptosis. Mol. Cell. Biochem. 174:185-188. | |
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| Samali, A., Cai, J., Zhivotovsky, B., Jones, D.P., and Orrenius, S. 1999. Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of Jurkat cells. EMBO J. 19:2040-2048. | |
| 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. | |
| Verhagen, A.M., Ekert, P.G., Pakusch, M., Silke, J., Connolly, L.M., Reid, G.E., Moritz, R.L., Simpson, R.J., and Vaux, D.L. 2000. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102:43-53. | |
| Zamzami, N., Susin, S.A., Marchetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M., and Kroemer, G. 1996. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183:1533-1544. | |
| Zhivotovsky, B., Samali, A., Gahm, A., and Orrenius, S. 1999. Caspases: Their intracellular localization and translocation during apoptosis. Cell Death Differ. 6:644-651. | |
| Zoratti, M. and Szabo, I. 1995. The mitochondrial permeability transition. Biochem. Biophys. Acta. 121:139-176. | |
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