This work was supported by the National Institute of Aging (AG04418), National Institute on Drug Abuse, National Institute of Health-Karolinska Institutet Graduate Partnerships Program, Karolinska Institutet, Swedish Research Council, Swedish Brain Power, and Swedish Brain Foundation. Many thanks to Mattias Karlen and Dr. Giuseppe Coppotelli for creative support with Figure 1 and 2, respectively; Karin Pernold for technical assistance; and Drs. Barry J. Hoffer, Lars Olson, and Nils-G&ouml;ran Larsson for much helpful advice and discussion.

1. Tissue preparation for cryosectioning
<ol>
	<li>Sacrifice the animal by either cervical dislocation or decapitation, in accordance with available ethical permit.</li>
	<li>Quickly collect tissues of interest (e.g. brain), and rapidly freeze on dry ice (tissues may require freezing in isopentane or propane chilled with liquid nitrogen to obtain optimal morphology). Store tissues in aluminum foil at -80 &deg;C until ready to section.</li>
	<li>Embed frozen tissue in preparation for cryosectioning.</li>
	<li>Collect 14-&mu;m cryostat sections at -21 &deg;C (may need to adjust temperature &plusmn; 1-2 &deg;C). Thaw sections onto slides using heating block, and store slides without coverslipping at -20 &deg;C until ready to use.</li>
</ol>
2. COX histochemistry
<ol>
	<li>Allow slides to dry at room temperature for 1 hour. Put slides in a slide-staining chamber with wet filter paper, cut into strips. To obtain consistent results in each experiment, it is recommended to process a maximum of ten slides per experiment to minimize time delays.</li>
	<li>Prepare under a chemical hood 1X DAB, 100 &mu;M cytochrome c in 0.1 M PBS pH=7.0. Vortex quickly.</li>
	<li>Add 2 &mu;g bovine catalase (2 &mu;g ml-1 or approximately 4 IU ml-1). Mix well by vortexing to break up all the grains of catalase.</li>
	<li>Apply 150- 200 &mu;L of incubation medium to each slide, use pipette tip to spread evenly onto all sections.</li>
	<li>Incubate the slides for 40 minutes at 37 &deg;C.</li>
	<li>Remove excess solution from the slides. Wash slides 4 times, 10 minutes each time, in 0.1 M PBS pH=7.0.</li>
	<li>Return slides to slide-staining chamber with wet paper strips.</li>
</ol>
3. SDH histochemistry
<ol>
	<li>Prepare under a chemical hood 1.5 mM NBT, 130 mM sodium succinate, 0.2 mM PMS, and 1.0 mM sodium azide in 0.1 M PBS pH=7.0. Take caution to shield the PMS from light. Vortex quickly.</li>
	<li>Apply 150-200 &mu;L of incubation medium to each slide, use pipette tip to spread evenly onto all sections.</li>
	<li>Incubate the slides for 40 minutes at 37 &deg;C.</li>
	<li>Remove excess solution from the slides. Wash slides 4 times, 10 minutes each time, in 0.1 M PBS pH=7.0.</li>
	<li>Dehydrate the slides for 2 minutes in the following concentrations of ethanol: 70%, 70%, 95%, 95%, 99.5%. Then allow 10 minutes in an additional 99.5% step.</li>
	<li>Place slides in xylene for 10 minutes. Mount with Entellan and coverslip. Allow the slides to dry overnight, or at least 1-2 hours in a ventilated area.</li>
</ol>
4. Determination of mitochondrial dysfunction
<ol>
	<li>The amount of mitochondrial dysfunction is indicated by the amount of cellular blue staining. To semi-quantify these amounts, slides should be coded and visualized under bright-field microscopy. Semi-quantification should be performed on a blind basis using a scale, for example from 0-4 (0, no blue staining; 4, only blue staining). It is best to perform this kind of semi-quantification on several sections from a given subject/animal to calculate a mean value for each subject/animal.</li>
	<li>Statistics should be performed using a non-parametric test, such as Mann-Whitney or Kruskal-Wallis.</li>
</ol>
5. Appropriate specificity controls
<ol>
	<li>For specificity controls for COX activity, repeat "COX histochemistry" steps, and add 2.5 mM sodium azide, a terminal respiratory chain inhibitor.</li>
	<li>For specificity controls for SDH activity, repeat "SDH histochemistry" steps with the removal of sodium succinate and the addition of 50 mM malonate, a competitive inhibitor of SDH.</li>
	<li>Wash and dehydrate sections in an ethanol series, and then mount and coverslip the slides as described in steps 3.4 - 3.6.</li>
</ol>
6. Representative Results:
The overall scheme of the COX/SDH double-labeling histochemical assay is illustrated in Figure 2. Representative examples of appropriate COX/SDH double-labeling histochemistry in brain sections from wild-type and prematurely aging mtDNA mutator mice are shown in Figure 3. The dark brown staining in wild-type mice (Figure 3, left panel) showed normal COX activity. Cells with respiratory chain deficiencies, indicated by the blue staining, were revealed in 12 week-old mtDNA mutator mice, and these deficiencies became more widespread as mtDNA mutator mice aged to 46 weeks (Figure 3, center and right panel).
Examples of inappropriate COX/SDH double-labeling in brain sections from wild-type mice due to insufficient COX labeling are shown in Figure 4. Inadequate incubation time for the demonstration of COX activity, or reducing the availability of molecular oxygen by coverslipping the slide during incubation, resulted in a reduced deposition of the DAB reaction product, and thus allowed for formation of the blue formazan end product during the SDH incubation.
COX and SDH activities can also be investigated separately (Figure 5, left and center); however, the sequential labeling is helpful in identifying cells with COX deficiencies, due to the formation of the blue precipitate during the SDH incubation (Figure 3, center and right). Specificity controls for COX and SDH activities can also be done (Figure 5, right).
<img alt="Figure 1" src="/files/ftp_upload/3266/3266fig1.jpg" /> 
Figure 1. Mitochondrial respiratory Complexes I-V. The mitochondrial respiratory chain is located within the inner membrane and includes five complexes. The purpose of the respiratory chain is to transport electrons from Complex I to IV and in doing so it creates a proton gradient across the inner membrane used by Complex V (ATPase) to produce ATP. Red hexagons represent subunits encoded by mtDNA. White hexagons represent subunits encoded by nuclear DNA (note that Complex II is completely encoded from the nuclear genome). Thus, mutations in the mitochondrial genome could cause dysfunction of the respiratory chain due to mutations in the subunits of the respiratory chain complexes.
<img alt="Figure 2" src="/files/ftp_upload/3266/3266fig2.jpg" /> 
Figure 2. Flow chart of the COX/SDH double-labeling histochemical assay. Dissect the organs of interest, rapidly freeze the tissues on dry ice, and store them at - 80 &deg;C. Collect cryostat sections and keep at - 20 &deg;C until use. Allow sections to air-dry at room temperature for 1 hour. Prepare the incubation medium for COX histochemistry, apply it to the slides, and incubate for 40 minutes at 37 &deg;C. Wash the sections in PBS 4 times for 10 minutes each wash. Prepare the incubation medium for SDH histochemistry, apply it to the slides, and again incubate for 40 minutes at 37 &deg;C. Wash the sections again in PBS, dehydrate in an ethanol series, and then mount and coverslip the slides. The COX/SDH double-labeled sections are ready to view under bright-field microscopy within 1-2 hours.
<img alt="Figure 3" src="/files/ftp_upload/3266/3266fig3.jpg" /> 
Figure 3. Representative examples of COX/SDH double-labeling. Brain sections from wild-type and prematurely aging mtDNA mutator mice were sequentially labeled for COX and SDH activities. (Scale bar: 200 &mu;m.) Normal COX activity (indicated by dark brown color) was shown in hippocampus from wild-type mice (left). COX deficiencies (indicated by blue color) were revealed in hippocampus from mtDNA mutator mice (center and right). There was a further decrease in COX activity by 46 weeks of age in mtDNA mutator mice, suggesting widespread exacerbation of respiratory chain dysfunction. The observed mitochondrial dysfunction in mtDNA mutator mice 12 is caused by high levels of mtDNA point mutations as well as increased levels of linear deletions 5.
<img alt="Figure 4" src="/files/ftp_upload/3266/3266fig4.jpg" /> 
Figure 4. Examples of inappropriate COX/SDH double-labeling. Brain sections from wild-type mice were sequentially labeled for COX and SDH activities. (Scale bar: 200 &mu;m.) Inadequate incubation times (10 and 25 minutes) for the demonstration of COX activity resulted in a reduced deposition of the brown DAB reaction product, compared to the 40-minute incubation time (left and center). The shortened incubation times allowed for the formation of the blue formazan end product during the SDH incubation, misleadingly suggesting the presence of cells with COX deficiencies. Coverslipping the slides during the COX incubation also resulted in inaccurate formation and deposition of the DAB reaction product (right).
<img alt="Figure 5" src="/files/ftp_upload/3266/3266fig5.jpg" /> 
Figure 5. Individual COX and SDH labeling and specificity control. Brain sections from wild-type mice were separately labeled for COX and SDH activities, indicated by the dark brown color and the blue color, respectively (left and center). Although COX and SDH activities can be individually labeled, the sequential labeling has proved to be advantageous in locating cells with mitochondrial dysfunction. An example of a specificity control for COX and SDH activities in brain from a wild-type mouse showed absence of labeling (right). (Scale bar: 200 &mu;m.)

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No conflicts of interest declared.

The combined COX/SDH histochemical method enables the visualization of cells with mitochondrial dysfunction. This technique, with early studies dating back to 1968, remains popular, with many considering it the "gold standard" for identifying mitochondrial diseases in patients14,19,26,27. It is now frequently used to investigate mtDNA mutation-driven aging and aging-related disorders 12,13,18,20,21,24. The COX/SDH double-labeling method is often used in parallel with other techniques to identify spe...

<table><tbody><tr><td>Dry Ice</td><td>AGA Gas AB</td><td>block form</td><td></td></tr><tr><td>Isopentane (2-methylbutane)</td><td>Sigma-Aldrich</td><td>277258 CAS: 78-78-4</td><td></td></tr><tr><td>Cyrostat embedding solution</td><td>Sakura Finetek</td><td>Tissue Tek 4583&lt;strong&gt;&lt;/strong&gt;</td><td></td></tr><tr><td>Cryostat</td><td>Microm International</td><td>Microm Model HM 500M</td><td></td></tr><tr><td>Slides</td><td>Thermo Fisher Scientific, Inc.</td><td>Super Frost Plus Menzel Gl&amp;#228;ser J1800AMWZ</td><td></td></tr><tr><td>Cover glasses Borosilicate glass</td><td>VWR international</td><td>16004-098</td><td>24 x 50 mm</td></tr><tr><td>Filter Paper</td><td>Munktell Filter AB</td><td>Quality: 1350 Article Number: 242 001</td><td>430 x 430 mm</td></tr><tr><td>3,3&amp;#8242;-diaminobenzidine tetrahydrochloride (DAB)</td><td>Sigma-Aldrich</td><td>Sigma Liquid Substrate System, D7304</td><td></td></tr><tr><td>Cytochrome &lt;em&gt;c&lt;/em&gt; (Type III, from equine heart)</td><td>Sigma-Aldrich</td><td>C2506 CAS: 9007-43-6</td><td></td></tr><tr><td>Bovine catalase (from liver)</td><td>Sigma-Aldrich</td><td>C9322 CAS: 9001-05-2</td><td></td></tr><tr><td>Nitroblue tetrazolium (NBT)</td><td>Sigma-Aldrich</td><td>N6876 CAS: 298-83-9</td><td></td></tr><tr><td>Sodium succinate</td><td>Sigma-Aldrich</td><td>S2378 CAS: 6106-21-4</td><td></td></tr><tr><td>Phenazine methosulfate (PMS)</td><td>Sigma-Aldrich</td><td>P9625 CAS: 299-11-6</td><td>PMS is light sensitive. Shield from light.</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032 CAS: 26628-22-8</td><td></td></tr><tr><td>Xylene</td><td>VWR international</td><td>EM-XX0060-4</td><td></td></tr><tr><td>Entellan</td><td>VWR international</td><td>100503-870</td><td></td></tr><tr><td>Malonate (Malonic acid)</td><td>Sigma-Aldrich</td><td>M1296 CAS: 141-82-2</td><td></td></tr></tbody></table>

visualization of mitochondrial respiratory function using cytochrome c oxidase / succinate dehydrogenase (cox/sdh) double-labeling histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Watch this Scientific Journal Video about Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry at JoVE.com

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

visualization-mitochondrial-respiratory-function-using-cytochrome-c

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

36.6K Views.  Karolinska Institutet. The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Video: Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Developmental Biology

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule synthesis. Genetic and functional adaptations of mitochondria to the tumor environment drive proliferative and metastatic potential. The advent of DNA and RNA sequencing removed critical barriers to the evaluation of genetic mediators of tumorigenesis. However, to date, methodological approaches to evaluate tumor mitochondrial function remain elusive and require technical proficiency limiting the feasibility, ultimately diminishing diagnostic and prognostic value in both experimental and clinical settings. Here, we&#160;outline a simple and rapid method to quantify rates of oxidative phosphorylation (OXPHOS) and electron transfer (ET) capacity in freshly excised solid tumor homogenates using high-resolution respirometry. The protocol can be reproducibly applied across species and tumor types as well as adapted to evaluate a diversity of mitochondrial ET pathways. Using this protocol, we demonstrate that mice bearing a luminal B mammary cancer exhibit defective nicotinamide adenine dinucleotide-linked respiration and reliance on succinate to generate adenosine triphosphate via OXPHOS.

We developed a practical protocol and analytical approach to evaluate mitochondrial oxidative phosphorylation and electron transfer capacity in fresh tumor homogenates. This protocol can be easily adapted to survey various mitochondrial functions that contribute to cancer initiation, progression, and treatment response.

Mitochondria are essential to the onset and progression of cancer through energy production, reactive oxygen species regulation, and macromolecule ...

Cancer Research

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Biochemistry

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

Stable Isotope Tracing & LC-MS Analysis to Measure DHODH and SDH Activities

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Author Spotlight: Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

Neuroscience

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

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Chapters in this video

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Analytical Determination of Mitochondrial Function of Excised Solid Tumor Homogenates

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays