isolation of enriched mitochondrial fractions from cell cultures and small animal tissue samples for gel activity assays

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Supercomplexes (SCs) are supramolecular associations between individual respiratory chain complexes1,2. Since the initial identification of SCs and the description of their composition by the group of Sch&#228;gger2,3, later confirmed by other groups, it was established that they contain respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively) in different stoichiometries. Two main populations of SCs can be defined, those containing CI (and either CIII alone or CIII and CIV) and with very high molecular weight (MW, starting ~1.5 MDa for the smaller SC: CI + CIII2) and those containing CIII and CIV but not CI, with much smaller size (such as CIII2 + CIV with ~680 kDa). These SCs coexist in the inner mitochondrial membrane with free complexes, also in different proportions. Thus, while CI is mostly found in its associated forms (that is, in SCs: ~80% in bovine heart and more than 90% in many human cell types)3, CIV is very abundant in its free form (more than 80% in bovine heart), with CIII showing a more balanced distribution (~40% in its more abundant free form, as a dimer, in bovine heart).
While their existence is now generally accepted, their precise role is still under debate4,5,6,7,8,9,10. According to the plasticity model, different proportions of SCs and individual complexes can exist depending on the cell type or the metabolic status1,7,11. This dynamic nature of the assembly would allow cells to regulate the use of available fuels and the efficiency of the oxidative phosphorylation (OXPHOS) system in response to environmental changes4,5,7. SCs could also contribute to control the reactive oxygen species generation rate and participate in the stabilization and turnover of individual complexes4,12,13,14. Modifications of the SC assembly status have been described in association with different physiological and pathological situations15,16 and with the aging process17.
Thus, changes in the SC patterns have been described in yeast depending on the carbon source used for growth2&#160;and in cultured mammalian cells when glucose is substituted by galactose4. Modifications have also been reported in mouse liver after fasting8 and in astrocytes when mitochondrial fatty acid oxidation is blocked18. In addition, a decrease or alterations in SCs and OXPHOS have been found in Barth syndrome19, heart failure20, several metabolic21 and neurological22,23,24 disorders, and different tumors25,26,27,28. Whether these alterations in SC assembly and levels are a primary cause or represent secondary effects in these pathological situations is still under investigation15,16. Different methodologies can give information about the assembly and function of SCs; these include activity measurements8,29, ultrastructural analysis30,31, and proteomics32,33. A useful alternative that is increasingly being employed and is the starting point for some of the previously mentioned methodologies is the direct determination of SC assembly status by Blue native (BN) electrophoresis developed for this purpose by Sch&#228;gger&#39;s group34,35.
This approach requires reproducible and efficient procedures to obtain and solubilize mitochondrial membranes and can be complemented by other techniques such as in-gel activity analysis (IGA), second-dimension electrophoresis, and western blot (WB). A limitation in the studies on SC dynamics by BN electrophoresis can be the amount of starting cells or tissue samples. We present a series of protocols for the analysis of SC assembly and function, adapted from Sch&#228;gger&#39;s group methods, that can be applied to fresh or frozen cell or tissue samples starting from as little as 20 mg of tissue.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples 

Our research is focused on better understanding mitochondrial biogenesis, in particular, the OXPHOS System organization and Regulation, and also on defining the role that mitochondria play in diseases from cancer to neurodegenerative disorders. Mitochondria perform multiple and essential roles in the cell, conditioning its whole metabolism and survival. We're slowly increasing our knowledge of mitochondrial complexity, and its contribution to cell adaptation mechanisms in health and disease.We have contributed to establish a new organization model for the OXPHOS system, the plasticity model that integrates previous proposals and assumes the coexistence of super complexes and free complexes in different proportions to optimize its function and allow cellular adaptations. Our protocol allows starting from small tissues or cell culture samples to obtain enough mitochondrial protein amounts to analyze super complexes assembly and status and function by blue native electrophoresis or other techniques. We can do this reproducibly in a short time with a simple equipment and relatively with low costs.

Isolation of Enriched Mitochondrial Fractions from Cell Cultures and Small Animal Tissue Samples for Gel Activity Assays

isolation-enriched-mitochondrial-fractions-from-cell-cultures-small

Research

JoVE Journal

Biochemistry

113 次观看.  University of Zaragoza. 首先，将填充的细胞沉淀重悬于等于细胞沉淀体积 7 倍的低渗缓冲液中。将细胞悬液转移到 Potter-Dounce 匀浆器中，在冰上孵育 2 分钟，让细胞膨胀。通过在均质器中执行 8 到 10 次冲程来打破细胞膜，该匀浆器与以 600 RPM 旋转的电机驱动的特氟龙杵相连。向细胞悬液中加入等体积的高渗缓冲液，以产生等渗环境。将匀浆转移到 10 至 15 毫升的试管中。在 4 摄氏度下以 1， 0...

视频: 从细胞培养物和小动物组织样品中分离富集的线粒体组分，用于凝胶活性测定

isolation of mitochondria for mitochondrial supercomplex analysis from small tissue and cell culture samples 

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

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analyzing mitochondrial supercomplexes in mammalian tissue fractions via in-gel activity assays

Analyzing Mitochondrial Supercomplexes in Mammalian Tissue Fractions via In-Gel Activity Assays

preparation of mitochondrial enriched fractions for metabolic analysis in drosophila

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

isolation of ca1 nuclear enriched fractions from hippocampal slices to study activity-dependent nuclear import of synapto-nuclear messenger proteins

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and...

isolation of viral replication compartment-enriched sub-nuclear fractions from adenovirus-infected normal human cells

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

robust mitochondrial isolation from rodent cardiac tissue 

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution...

Robust Mitochondrial Isolation from Rodent Cardiac Tissue 

isolation and functional assessment of human breast cancer stem cells from cell and tissue samples

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

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mitochondrial isolation and quality assessment in rodent cardiac tissue for optimizing respirometry assays

Mitochondrial Isolation and Quality Assessment in Rodent Cardiac Tissue for Optimizing Respirometry Assays

analyzing supercomplexes of the mitochondrial electron transport chain with native electrophoresis, in-gel assays, and electroelution

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions.  This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream...

fast and specific assessment of the halogenating peroxidase activity in leukocyte-enriched blood samples

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory...

method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic...

determination of mitochondrial respiration and glycolysis in ex vivo retinal tissue samples

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

derivation of enriched oligodendrocyte cultures and oligodendrocyte/neuron myelinating co-cultures from post-natal murine tissues

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons...

cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

Here, we describe a method for efficient cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures.  This simple protocol provides flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell...

magnetic isolation of microglial cells from neonate mouse for primary cell cultures

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures

Primary microglia cultures are commonly used to evaluate new anti-inflammatory molecules. The present protocol describes a reproducible and relevant method to magnetically isolate microglia from neonate...

mitochondrial isolation from skeletal muscle

Mitochondrial Isolation from Skeletal Muscle

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...

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...