Efficient Isolation of Pure and Functional Mitochondria from Mouse Tissues Using Automated Tissue Disruption and Enrichment with Anti-TOM22 Magnetic Beads

Andras Franko,Rudolf J Wiesner,Martin Hrabĕ De Angelis,Ian C D Johnston,Axel K Walch,Wolfgang Wurst,Stefanie M Hauck,Olivier R Baris,Michaela Aichler,Christine Von Toerne,Eva Bergschneider

doi:10.1371/journal.pone.0082392

Abstract

To better understand molecular mechanisms regulating changes in metabolism, as observed e.g. in diabetes or neuronal disorders, the function of mitochondria needs to be precisely determined. The usual isolation methods such as differential centrifugation result in isolates of highly variable quality and quantity. To fulfill the need of a reproducible isolation method from solid tissues, which is suitable to handle parallel samples simultaneously, we developed a protocol based on anti-TOM22 (translocase of outer mitochondrial membrane 22 homolog) antibody-coupled magnetic beads. To measure oxygen consumption rate in isolated mitochondria from various mouse tissues, a traditional Clark electrode and the high-throughput XF Extracellular Flux Analyzer were used. Furthermore, Western blots, transmission electron microscopic and proteomic studies were performed to analyze the purity and integrity of the mitochondrial preparations. Mitochondrial fractions isolated from liver, brain and skeletal muscle by anti-TOM22 magnetic beads showed oxygen consumption capacities comparable to previously reported values and little contamination with other organelles. The purity and quality of isolated mitochondria using anti-TOM22 magnetic beads was compared to traditional differential centrifugation protocol in liver and the results indicated an obvious advantage of the magnetic beads method compared to the traditional differential centrifugation technique.

Highlights

Mitochondrial performance needs to be investigated in many human disorders, as their altered function often contributes to or is at least suspected to play a role in the development of disease [1]
Since the common mitochondrial isolation methods do not necessarily selectively discriminate between mitochondria and other organelles such as endoplasmic reticulum (ER) and peroxisomes, we investigated those potential contaminations with organelle-specific antibodies
Western blots were labeled with an antibody against the protein disulfide isomerase A2 (PDI), which is localized in the ER [24], and against PEX1, a peroxisomal protein

Summary

Introduction

Mitochondrial performance needs to be investigated in many human disorders, as their altered function often contributes to or is at least suspected to play a role in the development of disease [1]. Mice appear to be an appropriate model for understanding the molecular mechanisms underlying such disorders, and exploring how mitochondrial function can affect tissues. Since one of the major functions of mitochondria is the production of ATP via oxidative phosphorylation, measurements of oxygen consumption using isolated organelles are commonly performed to identify potential mitochondrial dysfunction. New devices such as the XF Extracellular Flux Analyzer were developed to measure oxygen consumption in many samples simultaneously, including isolated mitochondria, allowing the exploration of mitochondrial function in smaller samples and in a highly parallel fashion compared to the traditional Clark electrode [9,10]. To match the convenience of such high-throughput devices for analyzing mitochondrial respiration in mouse tissues, there is a need for a standardized isolation method that allows simultaneous handling of numerous samples

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Conclusion