Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

Nature Protocols volume 3, pages 1228–1239 (2008)Cite this article

Abstract

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45–75 min and between 1 and 3 h for the mitochondrial isolation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2: Typical appearance of a centrifuge tube at the conclusion of the density gradient centrifugation step in Method A (Step 12A(ix)).

Similar content being viewed by others

References

  1. Schneider, W.C. & Hogeboom, G.H. Intracellular distribution of enzymes V. Further studies on the distribution of cytochrome c in rat liver homogenates. J. Biol. Chem. 183, 123–128 (1950).

    CAS  Google Scholar 

  2. Pallotti, F. & Lenaz, G. Isolation and subfractionation of mitochondria. Methods Cell Biol. 80, 3–44 (2007).

    Article  CAS  Google Scholar 

  3. Grey, E.G. & Whittaker, V.P. The isolation of nerve endings from brain: An electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96, 79–89 (1962).

    Google Scholar 

  4. Clark, J.B. & Nicklas, W.J. The metabolism of rat brain mitochondria. Preparation and characterization. J. Biol. Chem. 18, 4724–4731 (1970).

    Google Scholar 

  5. Stahl, W.L., Smith, J.C., Napolitano, L.M. & Basford, R.E. Brain mitochondria I. Isolation of brain mitochondria. J. Cell Biol. 19, 293–307 (1963).

    Article  CAS  Google Scholar 

  6. Sims, N.R. Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J. Neurochem. 55, 698–707 (1990).

    Article  CAS  Google Scholar 

  7. Salganicoff, L. & De Robertis, E. Subcellular distribution of the enzymes of the glutamic acid, glutamine and gamma-aminobutyric acid cycles in rat brain. J. Neurochem. 12, 287–309 (1965).

    Article  CAS  Google Scholar 

  8. Neidle, A., van den Berg, C.J. & Grynbaum, A. The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients. J. Neurochem. 16, 225–234 (1969).

    Article  CAS  Google Scholar 

  9. Tanaka, R. & Abood, L.G. Isolation from rat brain of mitochondria devoid of glytolytic activity. J. Neurochem. 10, 571–576 (1963).

    Article  CAS  Google Scholar 

  10. Lai, J.C.K. & Clark, J.B. Preparation of synaptic and non-synaptic mitochondria from mammalian brain. Methods Enzymol. 55, 51–60 (1979).

    Article  CAS  Google Scholar 

  11. Anderson, M.F. & Sims, N.R. Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res. Brain Res. Protoc. 5, 95–101 (2000).

    Article  CAS  Google Scholar 

  12. Elias, P.M., Goerke, J. & Friend, D.S. Freeze-fracture identification of sterol–digitonin complexes in cell and liposome membranes. J. Cell Biol. 78, 577–596 (1978).

    Article  CAS  Google Scholar 

  13. Colbeau, A., Nachbaur, J. & Vignais, P.M. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta Biomemb. 249, 462–492 (1971).

    Article  CAS  Google Scholar 

  14. Booth, R.F.G. & Clark, J.B. A method for the rapid separation of soluble and particulate components of rat brain synaptosomes. FEBS Lett. 107, 387–392 (1979).

    Article  CAS  Google Scholar 

  15. Greenawalt, J.W. The isolation of outer and inner mitochondrial membranes. Methods Enzymol. 31, 310–323 (1974).

    Article  CAS  Google Scholar 

  16. Brustovetsky, N., Jemmerson, R. & Dubinsky, J.M. Calcium-induced cytochrome c release from rat brain mitochondria is altered by digitonin. Neurosci Lett. 332, 91–94 (2002).

    Article  CAS  Google Scholar 

  17. Brown, M.R., Sullivan, P.G. & Geddes, J.W. Synaptic mitochondria are more susceptible to Ca2+ overload than nonsynaptic mitochondria. J. Biol. Chem. 281, 11658–11668 (2006).

    Article  CAS  Google Scholar 

  18. Lifshitz, J., Janmey, P.A. & McIntosh, T.K. Photon correlation spectroscopy of brain mitochondrial populations: Application to traumatic brain injury. Exp. Neurol. 197, 318–329 (2006).

    Article  CAS  Google Scholar 

  19. Morota, S. et al. Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition. J. Neurochem. 103, 2066–2076 (2007).

    Article  CAS  Google Scholar 

  20. Naga, K.K., Sullivan, P.G. & Geddes, J.W. High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition. J. Neurosci. 27, 7469–7475.

  21. Tsuchiya, M., Takahashi, M., Tomoda, M., Ueda, W. & Hirakawa, M. Halothane impairs the bioenergetic functions of isolated rat liver mitochondria. Toxicol. Appl. Pharmacol. 104, 466–475 (1990).

    Article  CAS  Google Scholar 

  22. Chávez, J.C., Pichiule, P., Boero, J. & Arregui, A. Reduced mitochondrial respiration in mouse cerebral cortex during chronic hypoxia. Neurosci. Lett. 193, 169–172 (1995).

    Article  Google Scholar 

  23. Oliveira, J.M.A. et al. Mitochondrial dysfunction in Hungtington's disease: the bioenergetics of isolated and in situ mitochondria from transgenic mice. J. Neurochem. 101, 241–249 (2007).

    Article  CAS  Google Scholar 

  24. Marini, S. et al. Expression, microsomal and mitochondrial activities of cytochrome P450 enzymes in brain regions from control and phenobarbital-treated rabbits. Life Sci. 80, 910–917 (2007).

    Article  CAS  Google Scholar 

  25. Ansari, M.A. et al. In vivo administration of D609 leads to protection of subsequently isolated gerbil brain mitochondria subjected to in vitro oxidative stress induced by amyloid beta-peptide and other oxidative stressors: Relevance to Alzheimer's disease and other oxidative stress-related neurodegenerative disorders. Free Radic. Biol. Med. 41, 1694–1703 (2006).

    Article  CAS  Google Scholar 

  26. Bhagwat, S.V., Boyd, M.R. & Ravindranath, V. Multiple forms of cytochrome P450 and associated monooxygenase activities in human brain mitochondria. Biochem. Pharmacol. 59, 573–582 (2000).

    Article  CAS  Google Scholar 

  27. Rajapaske, N., Shimizu, K., Payne, M. & Busija, D. Isolation and characterization of intact mitochondria from neonatal rat brain. Brain Res. Brain Res. Protoc. 8, 176–183 (2001).

    Article  Google Scholar 

  28. LaFrance, R., Brustovetsky, N., Sherburne, C., DeLong, D. & Dubinsky, J.M. Age-related changes in regional brain mitochondria from Fischer 344 rats. Aging Cell 4, 139–145 (2005).

    Article  CAS  Google Scholar 

  29. Shalbuyeva, N., Brustovetsky, T., Bolshakov, A. & Brustovetsky, N. Calcium-dependent spontaneously reversible remodelling of brain mitochondria. J. Biol. Chem. 281, 37547–37558 (2006).

    Article  CAS  Google Scholar 

  30. Kristian, T., Hopkins, I.B., McKenna, M.C. & Fiskum, G. Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation. J. Neurosci. Meth. 152, 136–143.

  31. Sims, N.R. Selective impairment of respiration in mitochondria isolated from brain subregions following transient forebrain ischemia in the rat. J. Neurochem. 56, 1836–1844 (1991).

    Article  CAS  Google Scholar 

  32. Sims, N.R. & Blass, J.P. Expression of classical mitochondrial respiratory responses in homogenates of rat forebrain. J. Neurochem. 47, 496–505 (1986).

    Article  CAS  Google Scholar 

  33. Johnson, M.K. The intracellular distribution of glycolytic and other enzymes in rat-brain homogenates and mitochondrial preparations. Biochem. J. 77, 610–618 (1960).

    Article  CAS  Google Scholar 

  34. Sims, N.R. & Carnegie, P.R. A rapid assay for 2′, 3′-cyclic nucleotide 3′-phosphohydrolase. J. Neurochem. 27, 769–772 (1976).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centre for Neuroscience and Department of Medical Biochemistry, School of Medicine, Flinders University, GPO Box 2100, Adelaide, 5001, South Australia, Australia

    Neil R Sims & Michelle F Anderson

Authors
  1. Neil R Sims
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michelle F Anderson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Neil R Sims.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sims, N., Anderson, M. Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 3, 1228–1239 (2008). https://doi.org/10.1038/nprot.2008.105

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2008.105

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing