We would like to acknowledge Daniel A. Beard and Kalyan C. Vinnakota for their foundational contributions to this protocol. This work was funded by NSF CAREER grant MCB-2237117.

The use and treatment of all vertebrate animals were performed in accordance with approved protocols reviewed and accepted by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University. This protocol was designed using both male and female Hartley albino guinea pigs and Sprague Dawley (SD) rats. For the isolation of cardiac mitochondria from guinea pigs, animals were sacrificed at ages between 4-6 weeks (300-450 g). Cardiac mitochondria from SD rats of both sexes were obtained between the ages of 10-13 weeks (250-400 g). Recipes for buffers are described in Table 1 and should be prepared in advance. Details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Experimental preparation
<ol>
	<li>Before starting the isolation, label two 50 mL centrifugation tubes as &#34;Spins 1 and 2&#34; and &#34;Spin 3&#34;.</li>
	<li>Place tubes, freshly thawed isolation buffer (IB), sharp mincing scissors, assembled homogenization probe, and a 5 mL beaker or equivalent-sized container on ice.</li>
	<li>Place freshly thawed respiration buffer (RB) in an incubator to warm for subsequent respiratory assays.</li>
	<li>Pre-chill a refrigerated centrifuge to 4 &#176;C.</li>
	<li>Ensure that all equipment is arranged and 20 &#181;L of 7-15 U/mg protease from Bacillus licheniformis has been added to the tube labeled &#34;Spins 1 and 2&#34;.</li>
	<li>Set up a gravity-dependent pressure system for perfusion via cannulation using Cardioplegia buffer (CB, Table 1), glass cannula, and plastic tubing with a stopcock valve attached.</li>
	<li>Arrange surgical tools and include proper scissors and forceps for dissection and cannulation of the heart. 
	NOTE: All water should be of pure quality (18.2 M&#937;&#183;cm)</li>
</ol>
2. Tissue dissection
<ol>
	<li>Inject animals with sterile heparin sulfate intraperitoneally at a dose of 500 U/kg.</li>
	<li>Allow the animal to sit in the induction chamber for 15 min after heparin administration. During this time, supply 2 L/min of pure O2 gas to fully oxygenate the animals, calm them, and minimize any stress that may have adverse consequences on tissue of interest.</li>
	<li>Start anesthesia induction by a continuous flow of isoflurane at 0.5%. After 1 min, increase to 1%. Continue toincrease by 1% every minute until 5% is reached. Once at 5%, wait for 1 min and monitor the animal&#39;s breathing pattern.</li>
	<li>Once breathing has slowed and becomes labored (approximately at the 6.5 min mark) turn off isoflurane and oxygen flow.</li>
	<li>Quickly remove the animal from the induction chamber and check for appropriate anesthetic depth by squeezing a paw and checking for the corneal reflex. If the animal responds to either stimulus, then place it back in the induction chamber, readminister anesthesia, and repeat the anesthetic depth check.</li>
	<li>Once the proper depth of anesthesia is reached, quickly decapitate with a guillotine to severe the cervical spine and place the prone body on ice.</li>
	<li>Make two parallel vertical incisions from the clavicles proceeding along the lateral rib cage down the length of the thorax. Ensure that the incisions are deep enough to cut through the ribs on the lateral thorax, but avoid damaging intrathoracic structures such as the heart or great vessels. 
	NOTE: Vertical incision sizes depend on the animal being used. If using guinea pigs and rats, cut to the diaphragm (approximately 6.35 cm for rats and 11 cm for guinea pigs). If using mice, perform a standard thoracotomy27.</li>
	<li>Expose the heart using a hemostat to displace the anterior thorax and pack the exposed thoracic cavity with ice. This step minimizes warm ischemia time and enhances the viability of the isolated organelles.</li>
	<li>Use tweezers to bluntly dissect the thymus and pericardium and fully expose the heart.</li>
	<li>Using forceps, apply gentle inferior traction on the heart to expose and identify the aorta. The aorta is the thickest great vessel branching out from the base of the heart. Other large vessels, such as the pulmonary vein, are noticeably more translucent than the aorta.</li>
	<li>Cut the aorta approximately 4-6 mm above the aortic root but below the carotid branching points.</li>
	<li>Cannulate the aorta and perfuse the heart28,29 with ice-cold cardioplegia (CB) solution using a gravity-dependent pressure head until the coronary arteries are cleared of blood and the organ appears blanched. 
	NOTE: For large rodents, a cannula diameter of 1.8-2.2 mm works well, while for smaller rodents, a diameter range of 1.4-1.8 mm is recommended. Retro-perfusion should take no more than 15-30 s for the coronary vessels to clear of blood.</li>
	<li>Isolate the ventricular myocardium by dissecting away the atria, cartilaginous valvular tissue, and fatty tissues.</li>
	<li>Place ventricles in a pre-chilled 10 mL beaker containing 0.1-0.2 mL ice-cold IB.</li>
	<li>Mince tissue with sharp surgical scissors until pieces are approximately 1 mm3.</li>
</ol>
3. Mitochondrial purification and protein quantification
<ol>
	<li>Transfer the minced tissue into the pre-chilled centrifuge tube labeled &#34;Spins 1 and 2&#34; containing the protease solution.</li>
	<li>Add ice-cold IB to a final volume of 25 mL.</li>
	<li>Using a motorized handheld rotor-stator homogenizer, disperse the tissue at 18,000 rpm on ice for 20-25 s.</li>
	<li>Centrifuge homogenized tissue in a tube labeled &#34;Spins 1 and 2&#34; at 8,000 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the supernatant (which contains protease) by pouring it into the waste carboy and gently rinse the pellet with 5 mL of IB to remove residual protease.</li>
	<li>After discarding the rinse, resuspend the pellet with fresh ice-cold IB to a final volume of 25 mL by gentle vortex.</li>
	<li>Centrifuge at 800 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant (contains mitochondria) by gently pouring it into a pre-chilled 50 mL tube labeled &#34;Spin 3&#34;. While pouring, take care to avoid dislodging the loose upper portion of the pellet. As an alternative, a transfer pipette or stripette can be used to collect the supernatant.</li>
	<li>Centrifuge the supernatant at 8000 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the resulting supernatant and retain the mitochondria-containing pellet.</li>
	<li>Use a lint-free wipe to absorb excess supernatant from the inside wall of the tube, taking care to avoid disturbing the pellet. Keep the pellet at 4 &#176;C on ice as much as possible.</li>
	<li>To resuspend the mitochondria, add 80 &#181;L of ice-cold IB to the bottom of the tube. Gently resuspend the pellet by repeatedly washing IB over the pellet.</li>
	<li>To avoid creating bubbles, set a micropipette to aspirate and deliver between 40-60 &#181;L of volume.</li>
	<li>As the mitochondrial pellet disperses, increase the micropipette volume to efficiently resuspend the pelleted mitochondria. Avoid touching the pellet with the pipette tip, and avoid making bubbles.</li>
	<li>Once resuspended, transfer the mitochondria to a pre-chilled microcentrifuge tube and label it as stock mitochondria. Make a note of the total resuspension volume.</li>
	<li>To determine the mitochondrial protein concentration in the sample, conduct a total protein assay using the well-known BCA or Bradford protein assays as defined by the manufacturer&#39;s instructions. 
	NOTE: An alternative or complementary strategy to assess yield is to determine the citrate synthase activity. For reference, mitochondrial content can be quantified by following the protocol described in Vinnakota et al.30.</li>
</ol>
4. Quality control checks
<ol>
	<li>In a pre-chilled microcentrifuge tube, dilute the mitochondrial stock to the desired working concentration with IB. 
	NOTE: Mitochondrial stocks are diluted to 40 mg/mL to work at 0.1 mg/mL final concentration for respirometry assays when using isolates from cardiac tissue.</li>
	<li>Rinse oxygraph chambers, stoppers, and microliter syringes ten times with distilled water to clean them before use in respirometry assays.</li>
	<li>To test the viability and quality of mitochondrial isolates, load 2.3 mL of respiration buffer (RB) into oxygraph chambers and allow the oxygen consumption signal to equilibrate at 37 &#176;C for about 10 min or when the rate is near 0.</li>
	<li>Once the signal is equilibrated, push down the stoppers and aspirate the excess buffer that emerges from the capillary of the stopper.</li>
	<li>Add 1 mM EGTA using a microliter syringe to chelate any residual calcium in the buffers or mitochondrial sample.</li>
	<li>To fuel respiration, add 5 mM sodium pyruvate and 1 mM L-malate.</li>
	<li>Following the addition of substrates, add a bolus of diluted mitochondria to achieve working concentration and allow respiration to occur for 5 min. This period is termed LEAK or State 2 respiration.</li>
	<li>At the 5 min mark, add a bolus of 500 &#181;M ADP to initiate State 3 respiration, also termed OXHPOS, and allow the mitochondria to respire until anoxia.</li>
	<li>Calculate the respiratory control ratio (RCR) by dividing the maximal rate of oxygen consumption during State 3 by the rate of oxygen consumption just before the addition of ADP in State 2 (see Figure 1). 
	NOTE: An alternative RCR expression of 1 - 1/RCR can also be used as a metric of quality, which is bounded between 0 and 1; however, it makes it difficult to differentiate quality using this metric when the RCR &#62; 10 (see Figure 2).</li>
	<li>Rinse chambers and stoppers 10 times with pure water to clean the oxygraph for subsequent assays. If respirometry is complete, fill chambers with 70% ethanol and place stoppers in chambers until the next use.</li>
</ol>

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

The authors declare that there is nothing to disclose.

Adhering to the methods concisely described in this protocol will ensure the isolation of well-coupled mitochondria from the cardiac tissue of small rodents, in addition to other tissue types and sources. Overall, the process should take a total of 3-3.5 h, during which all animal tissue, samples, and isolates should remain on ice at 4 &#176;C as much as possible to limit degradation. This procedure is robust and can be altered in several ways to better fit experimental goals and models utilized. One modulation that can ...

Mitochondria are subcellular organelles that establish cytoplasmic energetic conditions optimized for specific cell functions. While cellular, tissue, and organism-level studies can provide insights into mitochondrial function, isolating the organelles offers a level of experimental control not possible otherwise. Mitochondrial isolations have been performed since the 1940s, allowing mechanistic studies of metabolism and respiration across a variety of cells and tissues1,2. The historical relevance of mitochondria is also well-documented3. As the main producers of ATP, mitochondria play many key roles that are vital for optimal cellular and organ function4. Within the mitochondrial matrix, substrates are oxidized by the TCA cycle, producing reducing equivalents and mobile electron carriers such as NADH and UQH25,6. Cytochrome C is the third main mobile electron carrier in the mitochondrial biochemical reaction network7. These molecules are then oxidized by the transmembrane complexes of the electron transport system (ETS) embedded in the inner mitochondrial membrane8. Redox reactions of the ETS are coupled with proton translocation from the matrix to the intermembrane space. These processes establish an electrochemical proton gradient that is used to phosphorylate ADP with Pi by F1F0 ATP synthase to produce ATP9,10. The individual processes that occur at each complex can be explored with high-resolution respirometry using Clark-type electrodes or microplate oxygen consumption assays11,12. Additionally, disease models and treatments using isolated mitochondria can determine the impact or importance of mitochondrial function in the progression of certain pathologies. This has proven fruitful in the field of cardiology, where alterations in fuel and substrate delivery have been used to elucidate how mitochondrial dysfunction influences heart failure13,14,15,16. Mitochondria are also known to impact the development of other disease states such as diabetes, cancer, obesity, neurological disorders, and myopathies17,18. Therefore, the use of isolated or purified mitochondria enables mechanistic investigations of oxidative metabolism and ATP production in the source tissue.
There is no shortage of mitochondrial isolation protocols due to their importance in bioenergetic research. Additionally, highly specific methods tailored to subpopulations of mitochondria within tissues and cells can be found19,20. The basic procedural steps are similar between isolation methods, but variations can be made to buffer composition, homogenization steps, and centrifugation spins to improve the amount and quality of mitochondria. Changes to these aspects are based on the metabolic demand of the tissue, overall organ function, mitochondrial density, and other factors. In tissues such as the liver and skeletal muscle, handheld homogenizers are used to preserve mitochondrial integrity and limit damage to the mitochondrial membranes21. However, when isolating from kidneys, some protocols suggest using manually driven homogenization or commercial kits to yield better results22. Although both methods yield functional mitochondria, the quality of the organelles can become compromised by the additional time it takes to complete isolations using these protocols. Centrifugation is also vital to the extraction of mitochondrial protein, as it separates cellular components such as nuclei and other organelles from mitochondria23. During the isolation process, it is debated whether differential or density-based centrifugation should be implemented to obtain purer isolates24. While density centrifugation can separate mitochondria from organelles of similar specific gravity, such as peroxisomes, it is not well-established if mitochondria from these methods better represent in situ organelle function compared to mitochondria isolated using differential centrifugation. In the field of mitochondrial physiology, density-based centrifugation is preferred and can be easily altered to increase isolate purity. Whether changes to g-forces, centrifugation duration, and the number of centrifugation spins are incorporated should be considered before experimentation due to their influence on mitochondrial purification. Furthermore, mitochondrial resuspension, arguably the most crucial step during isolation, differs greatly between studies, with the use of scraping, vortex-based mixing, or homogenization23,25,26. Mechanical resuspension of these types can be too abrasive and impair the membrane integrity of mitochondria. For this reason, gentle washing should be performed to correct this. Despite the plethora of modulations and suggested methodologies, there are fewer comprehensive protocols with high reproducibility and adaptability for rodent models.
The methods described herein outline a detailed, robust, and highly reproducible protocol that yields purified, well-coupled mitochondria from small animal cardiac tissue. As demonstrated, this method can be easily adapted to accommodate the specific needs of each experiment and/or laboratory environment for isolating mitochondria from kidneys, liver, and cultured cells. Further modifications can be made to isolate mitochondria from tissues and other animals not listed here. Buffer recipes used for all isolations are provided and can be modified if needed. Similar to other published protocols, motorized homogenization, and differential centrifugation are implemented; however, adjustments are made to both the shearing time and the force at which the samples are centrifuged to consistently deliver high-quality mitochondrial isolates, depending on the isolation source. Notably, this protocol differs from others by using gentle washing via pipetting to resuspend pelleted mitochondria, which helps preserve mitochondrial membrane integrity and maintain the overall functionality of the organelles. Mitochondrial protein is quantified either by total protein determination or by measuring citrate synthase activity. The utility and broad applicability of this isolation method are further supported by the values of respiratory control ratios (RCR) achieved across various organisms and tissue sources.

<table><tbody><tr><td>1.7 mL microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>10 mL glass beaker</td><td></td><td></td><td>For organ disection and mincing</td></tr><tr><td>50 mL centrifuge tubes</td><td></td><td></td><td>Centrifugation</td></tr><tr><td>Adenosine 5&#039;-diphosphate monopotassium salt dihydrate</td><td>Sigma&amp;nbsp;</td><td>A5285</td><td>Respirometry assays</td></tr><tr><td>BSA Protein Assay Kit</td><td>Thermo Scentific</td><td>PI23225</td><td>Mitochondrial protein quantification</td></tr><tr><td>Dextrose</td><td>Sigma&amp;nbsp;</td><td>DX0145</td><td>For buffer (CB)</td></tr><tr><td>Ethylene glycol-bis(2-amino-ethylether)-N,N,N&#039;,N&#039;-tetraacetic acid&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>E4378</td><td>For buffers (CB and IB) and respirometry assays</td></tr><tr><td>Glass cannula</td><td>Radnoti</td><td></td><td>Guinea pig and rat heart perfusion</td></tr><tr><td>Heparin sodium porcine mucosa</td><td>Sigma&amp;nbsp;</td><td>SRE0027-250KU</td><td>Animal IP injection</td></tr><tr><td>High-resolution respirometer</td><td></td><td></td><td>Clark-type electrode; oxygraph with 2 mL chambers</td></tr><tr><td>Induction chamber</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Sigma&amp;nbsp;</td><td>792632</td><td>Anesthetic&amp;nbsp;</td></tr><tr><td>L-malic acid</td><td>Sigma&amp;nbsp;</td><td>02288-50G</td><td>Respirometry assays</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma&amp;nbsp;</td><td>M9272</td><td>For buffer (RB)</td></tr><tr><td>Mannitol</td><td>Sigma&amp;nbsp;</td><td>MX0214</td><td>For buffer (IB)</td></tr><tr><td>Microliter syringes</td><td></td><td></td><td>Sizes ranging from 5&amp;ndash;50 &amp;micro;L</td></tr><tr><td>Microplate reader</td><td></td><td></td><td>Must be able to incubate at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>MOPS</td><td>Sigma&amp;nbsp;</td><td>475898</td><td>For all buffers</td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; tank</td><td></td><td></td><td></td></tr><tr><td>OMNI THQ Homogenizer</td><td>OMNI International&amp;nbsp;</td><td>12-500</td><td>Similar rotor stator homogenizers will work</td></tr><tr><td>pipettes</td><td></td><td></td><td>Volumes of&amp;nbsp; 2&amp;ndash;20 &amp;micro;L; 20&amp;ndash;200 &amp;micro;L; 200&amp;ndash;1000 &amp;micro;L</td></tr><tr><td>Potassium chloride</td><td>Sigma&amp;nbsp;</td><td>P3911</td><td>For buffers (RB and CB)</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma&amp;nbsp;</td><td>795496</td><td>For buffers (IB and RB)</td></tr><tr><td>Protease from Bacillus licheniformis</td><td>Sigma&amp;nbsp;</td><td>P5459</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma&amp;nbsp;</td><td>S9888</td><td>For buffer (CB)</td></tr><tr><td>Sodium pyruvate</td><td>Fisher bioreagents</td><td>BP356-100</td><td>Respirometry assays</td></tr><tr><td>Sucrose</td><td>Sigma&amp;nbsp;</td><td>8510-OP</td><td>For buffer (IB)</td></tr><tr><td>Surgical dissection kit</td><td></td><td></td><td>Depends on animal and tissue source</td></tr><tr><td>Tabletop centrifuge</td><td></td><td></td><td>Must cool to 4 &amp;deg;C&amp;nbsp;</td></tr></tbody></table>

robust mitochondrial isolation from rodent cardiac tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Upon completion of mitochondrial isolation, the quality and functionality of the isolates should be tested via quantifying rates of oxygen consumption (JO2) using high-resolution respirometry. To do so, mitochondrial stocks were diluted to 40 mg/mL to allow for working concentrations of 0.1 mg/mL in 2 mL of RB for all respirometry assays using isolated cardiac mitochondria. Respiration was fueled by 5 mM sodium pyruvate and 1 mM L-malate in the presence of 1 mM EGTA, a calcium chelator, and was allowe...

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 respirometry.

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

robust-mitochondrial-isolation-from-rodent-cardiac-tissue

Research

JoVE Journal

Biochemistry

639 Views.  Michigan State University. Mitochondriën betrokken bij veel ziektepathologieën. Het zijn essentiële organellen die fungeren als oplaadbare en hernieuwbare biologische batterijen. Ze worden vaak de krachtpatser van de cel genoemd.Ze maken complex leven mogelijk. Ons lab is vooral geïnteresseerd in hoe deze biologische batterijen werken tijdens gezonde en ziektetoestanden om effectieve curatieven te ontdekken en nieuwe therapieën te ontwikkelen. Ons laboratorium heeft de aanw...

Video: Auteur in de schijnwerpers: de rol van mitochondriaal calciumfosfaat bij hartfalen en bio-energetica blootleggen

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Biology

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

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.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

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

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.

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

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 ...

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 ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

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