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Updated: May 28, 2026
Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue
Published on: March 23, 2015
Isolation of brain mitochondria from neonatal mice
Xiaoyang Wang1, Anna-Lena Leverin, Wei Han
1Perinatal Center, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden. xiaoyang.wang@fysiologi.gu.se
Abstract:
Mitochondria are key contributors to many forms of cell death including those resulting from neonatal hypoxic-ischemic brain injury. Mice have become increasingly popular in studies of brain injury, but there are few reports evaluating mitochondrial isolation procedures for the neonatal mouse brain. Using evaluation of respiratory activity, marker enzymes, western blotting and electron microscopy, we have compared a previously published procedure for isolating mitochondria from neonatal mouse brain (method A) with procedures adapted from those for adult rats (method B) and neonatal rats (method C). All three procedures use Percoll density gradient centrifugation as a key step in the isolation but differ in many aspects of the fractionation procedure and the solutions used during fractionation. Methods A and B both produced highly enriched fractions of well-coupled mitochondria with high rates of respiratory activity. The fraction from method C exhibited less preservation of respiratory properties and was more contaminated with other subcellular components. Method A offers the advantage of being more rapid and producing larger mitochondrial yields making it useful for routine applications. However, method B produced mitochondria that were less contaminated with synaptosomes and associated cytosolic components that suits studies that have a requirement for higher mitochondrial purification.
Insights
Researchers compared three methods for isolating mitochondria from neonatal mouse brains to study cell death. Methods A and B yielded high-quality mitochondria, with Method A being faster and Method B offering higher purity for specific research needs.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Mitochondria play a crucial role in cell death pathways, particularly in neonatal hypoxic-ischemic brain injury.
- Neonatal mouse models are increasingly used for brain injury research, but standardized mitochondrial isolation protocols are lacking.
Purpose of the Study:
- To evaluate and compare three distinct mitochondrial isolation procedures for the neonatal mouse brain.
- To determine the optimal method for obtaining high-purity, functional mitochondria for research.
Main Methods:
- Comparison of three isolation procedures (Method A: neonatal mouse brain, Method B: adult rat adapted, Method C: neonatal rat adapted).
- Evaluation techniques included respiratory activity assays, marker enzyme analysis, western blotting, and electron microscopy.
- All methods utilized Percoll density gradient centrifugation.
Main Results:
- Methods A and B yielded highly enriched, well-coupled mitochondria with preserved respiratory activity.
- Method C resulted in mitochondria with diminished respiratory function and increased contamination.
- Method A provided higher yields and was faster; Method B offered superior purity, with fewer synaptosome contaminants.
Conclusions:
- Methods A and B are suitable for isolating functional mitochondria from neonatal mouse brains, each with specific advantages.
- Method A is recommended for routine applications due to speed and yield.
- Method B is preferred for studies requiring highly purified mitochondria, minimizing synaptosome contamination.
