Mitochondrial Isolation
Mitochondrial Isolation - Related Videos
09:45
Mitochondrial Isolation from Skeletal Muscle
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Cited by 47 •
2011
This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.
05:09
Mitochondrial Isolation: A Technique to Isolate Mitochondria from Human Ovarian Cancer Tissue Sample
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2023
This video describes the procedure for isolating mitochondria from human ovarian cancer tissues using differential-speed centrifugation in combination with density gradient centrifugation. The isolated mitochondria can be used for proteomic analysis of human ovarian cancer mitochondrial proteome.
08:40
An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes
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Cited by 38 •
2016
The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.
05:53
Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay
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Cited by 11 •
2018
This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.
03:13
Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria
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2025
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.
FAQs
Differential centrifugation applies sequential centrifugal forces so cellular components separate according to differences in size and density. Larger structures such as nuclei can be separated from smaller mitochondrial fractions, while membranes and cytosolic material remain distinguishable in later fractions. This staged separation is central to obtaining mitochondria suitable for independent structural, functional, and biochemical studies.
Controlled homogenization must break open cells while preserving the organelles being studied. Excessive mechanical disruption can compromise mitochondrial structure, whereas insufficient disruption may leave cells unopened and reduce recovery. The degree of homogenization therefore affects both the amount of mitochondrial material obtained and the quality of subsequent measurements of respiration, ATP production, membrane potential, or apoptosis-related activity.
Temperature, osmotic conditions, and handling intensity are key quality variables. Maintaining an appropriate temperature and osmotic environment helps preserve mitochondrial structure during processing, while gentle handling limits mechanical damage. If these conditions are poorly controlled, the resulting preparation may be less suitable for evaluating mitochondrial function or biochemical activity independently from other cellular components.
Isolated mitochondria allow investigators to examine cellular respiration, ATP production, membrane potential, metabolism, and apoptosis-related activity without the full complexity of an intact cell. Studying these properties in a separated preparation can help connect mitochondrial behavior with broader biological processes, including changes in energy production and cellular responses associated with disease or toxic exposure.
A typical workflow begins with controlled homogenization to open cells or tissue while retaining organelles. The homogenate then undergoes sequential centrifugation steps, with centrifugal forces used to separate mitochondria from nuclei, membranes, cytosol, and other material. Appropriate temperature, osmotic conditions, and gentle handling are maintained throughout because processing conditions directly influence preparation quality.
The method is useful whenever researchers need to assess mitochondrial structure, function, or biochemical activity as an independent component of a biological sample. In physiology and disease research, it supports examination of respiration, energy production, metabolism, membrane potential, and apoptosis. Toxicology and drug-development studies can likewise use these measurements to evaluate mitochondrial effects of exposures or candidate treatments.