Abstract
Methods for isolating mitochondria from different rodent tissues have been established for decades. Although the general principles for crude mitochondrial preparations are largely shared across tissues – tissue disruption followed by differential centrifugation – critical differences exist for isolation from different tissues to optimize mitochondrial yield and function. This protocol offers a unified resource for preparations of isolated mitochondria from mouse liver, kidney, heart, brain, skeletal muscle, and brown and white adipose tissue suitable for functional analysis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Chappell JB, Hansford RG (1972) Preparation of mitochondria from animal tissues and yeasts. In: Birnie GD (ed) Subcellular components, 2nd edn. Butterworth-Heinemann, pp 77–91
Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435:297–312
Rogers GW, Brand MD, Petrosyan S, Ashok D, Elorza AA et al (2011) High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One 6:e21746
Divakaruni AS, Jastroch M (2022) A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements. Nat Metab 4:978–994
Toime LJ, Brand MD (2010) Uncoupling protein-3 lowers reactive oxygen species production in isolated mitochondria. Free Radic Biol Med 49:606–611
Wong HS, Monternier PA, Orr AL, Brand MD (2018) Plate-based measurement of superoxide and hydrogen peroxide production by isolated mitochondria. In: Palmeira C, Moreno A (eds) Mitochondrial bioenergetics, Methods in molecular biology, vol 1782, pp 287–299
Dröse S, Brandt U (2008) The mechanism of mitochondrial superoxide production by the cytochrome bc1 complex. J Biol Chem 283:21649–21654
Yang K, Doan MT, Stiles L, Divakaruni AS (2021) Measuring CPT-1-mediated respiration in permeabilized cells and isolated mitochondria. STAR Protoc 2:100687
Johnson D, Lardy H (1967) Isolation of liver or kidney mitochondria. Methods Enzymol 10:94–96
Liesa M, Luptak I, Qin F, Hyde BB, Sahin E et al (2011) The mitochondrial transporter ABC-me (ABCB10) is a novel gene required for cardiac recovery after ischemia-reperfusion. Circulation 124:806–813
Sahin E, Colla S, Liesa M, Moslehi J, Müller FL et al (2011) Telomere dysfunction induces metabolic and mitochondrial compromise. Nature 470:359–365
Woodall BP, Orogo AM, Najor RH, Cortez MQ, Moreno ER et al (2019) Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice. JCI Insight 5:e127713
Kushnareva Y, Murphy AN, Andreyev A (2002) Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation–reduction state. Biochem J 368:545–553
Cogswell AM, Stevens RJ, Hood DA (1993) Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions. Am J Phys 264:C383–C389
Lai N, Kummitha CM, Rosca MG, Fujioka H, Tandler B, Hoppel CL (2019) Isolation of mitochondrial subpopulations from skeletal muscle: optimizing recovery and preserving integrity. Acta Physiol (Oxf) 225:e13182
Benador IY, Veliova M, Mahdaviani K, Petcherski A, Wikstrom JD et al (2018) Mitochondria bound to lipid droplets have unique bioenergetics, composition, and dynamics that support lipid droplet expansion. Cell Metab 27:869–885.e6
Jones AE, Sheng L, Acevedo A, Veliova M, Shirihai OS et al (2021) Forces, fluxes, and fuels: tracking mitochondrial metabolism by integrating measurements of membrane potential, respiration, and metabolites. Am J Physiol Cell Physiol 320:C80–C91
Affourtit C, Quinlan CL, Brand MD (2012) Measurement of proton leak and electron leak in isolated mitochondria. In: Palmeira C, Moreno A (eds) Mitochondrial bioenergetics, Methods in molecular biology, vol 810, pp 165–182
Starkov AA (2010) Measurement of mitochondrial ROS production. In: Bross P, Gregersen N (eds) Protein Misfolding and cellular stress in disease and aging, Methods in molecular biology, vol 648, pp 245–255
Bhosale G, Duchen MR (2019) Investigating the mitochondrial permeability transition pore in disease phenotypes and drug screening. Curr Protoc Pharmacol 85:e59
Bertholet AM (2021) The use of the patch-clamp technique to study the thermogenic capacity of mitochondria. J Vis Exp 171. https://doi.org/10.3791/62618
Acin-Perez R, Benador IY, Petcherski A, Veliova M, Benavides GA et al (2020) A novel approach to measure mitochondrial respiration in frozen biological samples. EMBO J 39:e104073
Spinazzi M, Casarin A, Pertegato V et al (2012) Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc 7:1235–1246
Acín-Pérez R, Hernansanz-Agustín P, Enríquez JA (2020) Analyzing electron transport chain supercomplexes. Methods Cell Biol 155:181–197
Enríquez JA (2019) Mind your mouse strain. Nat Metab 1:5–7
Nedergaard J, Cannon B (1979) Overview--preparation and properties of mitochondria from different sources. Methods Enzymol 55:3–28
White AT, Philp A, Fridolfsson HN et al (2014) High-fat diet-induced impairment of skeletal muscle insulin sensitivity is not prevented by SIRT1 overexpression. Am J Physiol Endocrinol Metab 307:E764–E772
Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc 2:287–295
Heisler CR (1991) Mitochondria from rat liver: method for rapid preparation and study. Biochem Educ 19:35–38
Acknowledgments
We would like to acknowledge our colleagues and mentors Martin Brand, José Antonio Enríquez, Marc Liesa, Giovanni Manfredi, and Anne Murphy for their guidance over the years in helping us refine these protocols. This work was supported by NIH grant R35GM138003 (to A.S.D.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Acín-Pérez, R., Montales, K.P., Nguyen, K.B., Brownstein, A.J., Stiles, L., Divakaruni, A.S. (2023). Isolation of Mitochondria from Mouse Tissues for Functional Analysis. In: Papa, S., Bubici, C. (eds) Metabolic Reprogramming. Methods in Molecular Biology, vol 2675. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3247-5_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3247-5_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3246-8
Online ISBN: 978-1-0716-3247-5
eBook Packages: Springer Protocols