This study was supported by National Heart, Lung, and Blood Institute Grant HL- 103542, and The BCH Anesthesia Research Foundation&#8217;s Distinguished Trailblazer Award to CAP.

Preparation
<ol>
	<li>Prepare 1 M K-HEPES Stock Solution (adjust pH to 7.2 with KOH).</li>
	<li>Prepare 0.5 M K-EGTA Stock Solution (adjust pH to 8.0 with KOH).</li>
	<li>Prepare 1 M KH2PO4 Stock Solution.</li>
	<li>Prepare 1 M MgCl2 Stock Solution.</li>
	<li>Prepare Homogenizing Buffer (pH 7.2) 300 mM sucrose, 10 mM K-HEPES, and 1 mM K-EGTA. Store at 4 &#176;C.</li>
	<li>Prepare Respiration Buffer 250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2, 20 mM K-HEPES Buffer (pH 7.2) and 0.5 mM K-EGTA (pH 8.0). Store at 4 &#176;C.</li>
	<li>Prepare 10x PBS Stock Solution by dissolving 80 g of NaCl, 2 g of KCl, 14.4 g of Na2HPO4, and 2.4 g of KH2PO4 into 1 L double distilled H2O (pH 7.4).</li>
	<li>Prepare 1x PBS by pipetting 100 ml 10x PBS into 1 L double distilled H2O.</li>
	<li>Prepare Subtilisin A Stock by weighing out 4 mg of Subtilisin A into a 1.5 ml microfuge tube. Store at -20&#160;&#176;C until use.</li>
	<li>Prepare BSA Stock by weighing out 20 mg of BSA into a 1.5 ml microfuge tube. Store at -20&#160;&#176;C until use.</li>
</ol>
Mitochondrial Isolation (Figure 1)
<ol>
	<li>Immediately prior to isolation, dissolve Subtilisin A in 1 ml of Homogenizing Buffer.</li>
	<li>Immediately prior to isolation, dissolve BSA in 1 ml of Homogenizing Buffer.</li>
	<li>Collect two fresh tissue samples using a 6 mm biopsy sample punch and store in 1x PBS in a 50 ml conical centrifuge tube on ice.</li>
	<li>Transfer the two 6 mm punches of tissue to a dissociation C tube containing 5 ml of ice cold Homogenizing Buffer.</li>
	<li>Homogenize the tissue by fitting the dissociation C tube on the tissue dissociator and select the pre-set mitochondrial isolation cycle (60 sec homogenization).</li>
	<li>Remove the dissociation C tube to an ice bucket.</li>
	<li>Add 250 &#956;l of Subtilisin A Stock Solution to the homogenate, mix by inversion and incubate the homogenate on ice for 10 min.
	<ol>
		<li>Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.</li>
	</ol>
	</li>
	<li>Place a 40 &#181;m mesh filter onto a 50 ml conical centrifuge tube on ice and pre-wet the filter with Homogenizing Buffer and filter the homogenate into the 50 ml conical centrifuge tube on ice.</li>
	<li>Add 250 &#181;l of freshly prepared BSA Stock Solution to the filtrate and mix by inversion. 
	Note: Omit this step if mitochondrial protein determination is required.</li>
	<li>Place a 40 &#181;m filter onto a 50 ml conical centrifuge tube on ice and pre-wet the filter with Homogenizing Buffer and filter the homogenate into the 50 ml conical centrifuge tube on ice.</li>
	<li>Place a 10 &#181;m filter onto a 50 ml conical centrifuge tube on ice and pre-wet the filter with Homogenizing Buffer and filter the homogenate into the 50 ml conical centrifuge tube on ice.</li>
	<li>Transfer the filtrate to two pre-chilled 1.5 ml microfuge tubes and centrifuge at 9,000 x g for 10 min at 4 &#186;C.</li>
	<li>Remove supernatant and re-suspend and combine pellets in 1 ml of ice cold Respiration Buffer.</li>
</ol>
ATP Assay
Note: To determine the metabolic activity of isolated mitochondria an ATP luminescence assay can be performed using an ATP assay kit. The protocol, reagents and standards were supplied in the assay kit. A summary of the procedure is described below.
<ol>
	<li>Equilibrate kit reagents to RT.</li>
	<li>Prepare 10 mM ATP Stock Solution by dissolving lyophilized ATP pellet in 1,170 &#181;l of double distilled water. Store ATP standard Stock Solution and prepared mitochondrial samples on ice.</li>
	<li>Add 5 ml of Substrate Buffer solution to a vial of lyophilized substrate solution. Mix gently and place in the dark.</li>
	<li>Add 100 &#181;l of Respiration Buffer to all wells of&#160;a black, opaque bottom, 96 well plate.</li>
	<li>Add 10 &#181;l of mitochondria from the prepared samples to each well of a black, opaque bottom, 96 well plate. Note: Samples are plated in triplicate. Include a row for standards and three wells for the negative control (Respiration Buffer).</li>
	<li>Add 50 &#181;l of mammalian cell lysis solution to all wells, including standards and controls.</li>
	<li>Incubate the plate at 37 &#186;C for 5 min on an orbital shaker at 125 rpm.</li>
	<li>During the incubation prepare ATP standards in concentrations of 0.1 mM, 0.05 mM, 0.01 mM, 0.005 mM, 0.001 mM, and 0.0001 mM ATP from the 10 mM ATP Stock Solution. Store standards on ice.</li>
	<li>Following the incubation, add 10 &#181;l of ATP standards to corresponding wells as indicated on the plate map. Note: Standards are performed in duplicate.</li>
	<li>Add 50 &#181;l of the reconstituted substrate solution to each well.</li>
	<li>Incubate the plate at 37 &#186;C on the orbital shaker for 5 min at 125 rpm.</li>
	<li>Open Gen5 1.11 software on a computer linked with the spectrophotometer.</li>
	<li>Under &#8220;Create a New Item&#8221;, click on &#8220;Experiment&#8221;.</li>
	<li>Click on &#8220;Default Protocol&#8221;. Click &#8220;Ok&#8221;.</li>
	<li>In the left column, select &#8220;Protocol&#8221;, then &#8220;Procedure&#8221;.</li>
	<li>Select &#8220;Delay&#8221;. Set to 00:10:00. Click &#8220;Ok&#8221;.</li>
	<li>Select &#8220;Read&#8221;. Select &#8220;Luminescence&#8221; from drop down menu. Adjust other settings to the following: Read type Endpoint, Integration Time 0:01.00 MM:SS.ss, Filter Sets 1, Emission Hole, Optics Position Top, Sensitivity 100, Top Probe Vertical Offset 1.00 mm.</li>
	<li>When the plate is ready to be analyzed, click the &#8220;Read Plate&#8221; icon on the top row. Click &#8220;Read&#8221;. When the spectrophotometer opens, place the plate into the tray with well A1 in the upper left corner. A box with the temperature will open. Click &#8220;Read&#8221;. Note: Higher values correlate with increased ATP levels and higher metabolic activity.</li>
</ol>

<ol>
	<li>van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S. J., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondrial+factors+in+apoptosis:+a+Russian+roulette+with+more+than+one+bullet.">The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet.</a> Cell Death Differ. 9 (10), 1031-1042 (2002).</li><li>Szabadkai, G., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+the+hub+of+cellular+Ca2++signaling.">Mitochondria: the hub of cellular Ca2+ signaling.</a> Physiology (Bethesda). 23, 84-94 (2008).</li><li>Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+Dynamic+Organelles+in+Disease,+Aging,+and+Development.">Mitochondria: Dynamic Organelles in Disease, Aging, and Development.</a> Cell. 125 (7), 1241-1252 (2006).</li><li>Picard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+structure+and+function+are+disrupted+by+standard+Isolation+methods.">Mitochondrial structure and function are disrupted by standard Isolation methods.</a> PLoS ONE. 6 (3), e18317 (2011).</li><li>Bensley, R. R., Hoerr, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+cell+structure+by+freeze-drying+method;+preparation+and+properties+of+mitochondria.">Studies on cell structure by freeze-drying method; preparation and properties of mitochondria.</a> Anat Rec. 60, 449-455 (1934).</li><li>Claude, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+mammalian+liver+cells+by+differential+centrifugation+II.+Experimental+procedures+and+results.">Fractionation of mammalian liver cells by differential centrifugation II. Experimental procedures and results.</a> J Exp Med. 84 (1), 61-89 (1946).</li><li>Hogeboom, H., Schneider, W. C., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+mammalian+tissues;+isolation+of+intact+mitochondria+from+rat+liver;+some+biochemical+properties+of+mitochondria+and+submicroscopic+particulate+material.">Cytochemical studies of mammalian tissues; isolation of intact mitochondria from rat liver; some biochemical properties of mitochondria and submicroscopic particulate material.</a> J Biol Chem. 172 (2), 619-635 (1948).</li><li>Ernster, L., Schtaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+a+historical+Review.">Mitochondria: a historical Review.</a> J Cell Biol. 91 (3 Pt 2), 227s-255s (1981).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Schmitt, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+semi-automated+method+for+isolating+functionally+intact+mitochondria+from+cultured+cells+and+tissue+biopsies.">A semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies.</a> Anal Biochem. 443 (1), 66-74 (2013).</li><li>Fern&#225;ndez-Vizarra, E., Ferr&#237;n, G., P&#233;rez-Martos, A., Fern&#225;ndez-Silva, P., Zeviani, M., Enr&#237;quez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+for+biogenetical+studies:+An+update.">Isolation of mitochondria for biogenetical studies: An update.</a> Mitochondrion. 10 (3), 253-262 (2010).</li><li>Graham, J. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+isolated+mitochondria+during+early+reperfusion+for+cardioprotection.">Injection of isolated mitochondria during early reperfusion for cardioprotection.</a> American J Physiol Heart and Circ Physiol. 296 (1), 94-105 (2009).</li><li>Olson, M. S., Von Korff, R. 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All animals were treated in accordance with current institutional guidelines. We have no competing financial interests to disclose.

To successfully isolate mitochondria using this protocol it is essential to keep all solutions and tissue samples on ice to preserve mitochondrial viability. Even when maintained on ice, isolated mitochondria will exhibit a decrease in functional activity over time19. We recommend that all solutions and additions be pre-prepared. We pre-weigh and store Subtilisin A in 4 mg aliquots in 1.5 ml microfuge tubes and store them at -20 &#176;C. Similarly BSA is pre-weighed and stored in 20 mg aliquots in 1.5 ml micro...

A correction was made to: <a href="http://jove.unicartagenaproxy.elogim.com/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

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

Mitochondria exist in every cell in the body except red blood cells and are involved in a large number of important cellular and metabolic processes1-4. Because of these many functions, mitochondrial damage can have detrimental effects3. In order to investigate mitochondrial function and dysfunction several mitochondrial isolation methods have been described. The earliest published accounts of mitochondrial isolation date to the 1940s5-8. The first documented attempt demonstrated mitochondrial isolation by grinding liver tissue in a mortar followed by centrifugation in a salt solution at low speed5,8. Later, other groups expanded upon the original procedure and demonstrated tissue fractionation based on differential centrifugation6-8. These early methods formed the basis of current techniques which often incorporate homogenization, and/or differential centrifugation9-15. The number of homogenization and centrifugation steps varies among protocols. These repetitive steps increase the time for mitochondrial isolation and ultimately reduce viability. In addition, manual homogenization can cause mitochondrial damage and inconsistent results if not properly controlled10,16.
Recently, we used homogenization and differential centrifugation to isolate mitochondria for transplantation into myocardial tissue17,18. This lengthy isolation procedure required approximately 90 min and the clinical applicability of this method was therefore limited. To allow for acute therapeutic use in clinical and surgical treatment we have developed a rapid mitochondrial isolation procedure that can be performed in less than 30 min.
The major benefits of this protocol are that standardized tissue dissociation allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. In addition, the use of differential filtration in place of differential centrifugation eliminates time consuming and repetitive centrifugation steps allowing for more rapid isolation of highly purified, viable and respiration competent mitochondria.
The ability to isolate viable and respiration competent mitochondria in less than 30 min allows for clinical applicability. This isolation protocol has potential for use in coronary artery bypass grafting surgery (CABG) and other therapeutic procedures.

<table><tbody><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>84100</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H4034</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>Substilsin A</td><td>Sigma Aldrich</td><td>P5380</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>P5379</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>M8266</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S6191</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P2173</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374</td><td></td></tr><tr><td>ATPlite Luminescence Assay System,&lt;br/&gt; 1,000 Assay Kit</td><td>Perkin Elmer</td><td>6016941</td><td></td></tr><tr><td>50 ml Conical Tubes</td><td>BD</td><td>352098</td><td></td></tr><tr><td>40 &amp;mu;m Nylon Filters</td><td>BD&amp;nbsp;</td><td>352340</td><td></td></tr><tr><td>GentleMACS C tube</td><td>Miltenyl Biotech</td><td>120-005-331</td><td></td></tr><tr><td>1.5 ml Eppendorf tube</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>6 mm biopsy punch</td><td>Miltex</td><td>33-36</td><td></td></tr><tr><td>10 &amp;mu;m Pluristrainer</td><td>Pluriselect</td><td>43-500-10-03</td><td></td></tr><tr><td>Eppendorf Centrifuge 5415C</td><td>Marshall Scientific</td><td>EP-5415C&amp;nbsp;</td><td></td></tr><tr><td>GentleMACS Dissociator&amp;nbsp;</td><td>Miltenyl Biotech</td><td>130-093-235</td><td></td></tr><tr><td>96-well plates, tissue culture treated</td><td>VWR</td><td>82050-732</td><td></td></tr><tr><td>Rotomax 120 orbital shaker</td><td>Heidolph</td><td>544-41200-00</td><td></td></tr><tr><td>Synergy H4 Hybrid Multi-Mode&lt;br/&gt; &amp;nbsp;Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>Multisizer 4 Coulter Counter</td><td>Beckman Coulter</td><td>A63076</td><td></td></tr><tr><td>Oxytherm System</td><td>Hansatech Instruments</td><td></td><td></td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>267110</td><td></td></tr></tbody></table>

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 figure outlining the procedural steps in the isolation of mitochondria using tissue dissociation and differential filtration is shown in Figure 1. Total procedural time is less than 30 min.
Tissue samples were obtained using a 6 mm biopsy punch. Tissue weight was 0.18 &#177; 0.04 g (wet weight). The number of mitochondria isolated as determined by particle size counting was 2.4 x 1010 &#177; 0.1 x 1010 mitochondria for skeletal muscle and 2.75 x 10...

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

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

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 ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

38.0K Views.  Beth Israel Deaconess Medical Center and Harvard Medical School. 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 

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

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

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

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, 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 ...

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 ...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

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.

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.

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

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 ...

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.