We would like to thank Dr. Marc Liesa, Boston University School of Medicine, helpful discussions; Ms. Karin Murphy, Ms. Heather Gregory, and Mr. John Stone, all from Wake Forest School of Medicine, for helpful technical assistance in the development of this protocol.

NOTE: The protocol described was approved by the Institutional Review Board of Wake Forest School of Medicine. Informed consent was obtained in writing. All participants were healthy older adults (65-79 years) of both genders, with BMIs ranging from 23-35.

1. Skeletal Muscle Biopsy
<ol>
	<li>As previously described, 9 perform all biopsies in the early morning after an O/N fast. Ask the subjects to refrain from taking aspirin, prescription and over-the-counter non-steroidal anti-inflammatory drugs, or other compounds that may affect bleeding, platelets, or bruising for the week prior to the biopsy. Ask the participants to also refrain from any strenuous activity for at least 36 hr&#160;prior to the biopsy. 
	NOTE: Muscle is obtained from the Vastus lateralis using the percutaneous needle biopsy technique with a percutaneous needle under local anesthesia with 1% lidocaine. No medical complications or other reported adverse events from the procedure have occurred in our clinical research unit.</li>
	<li>NOTE: The biopsy procedure described is adapted from that of Bergstrom10.
	<ol>
		<li>Briefly, locally administer 1% lidocaine taking care not to infiltrate the muscle. Follow this with a 10&#160;min&#160;wait period to permit sufficient numbing.</li>
		<li>Take the biopsies from the belly of the muscle (the middle region of muscle between insertion and origin) avoiding subfascial and myotendonous areas.
		<ol>
			<li>Use percutaneous needle (a suction assisted reusable device with a side cutting window and inner cutting cylinder) and follow a fascial &#8220;pop&#8221; or resistance of the fascia as a guide.&#160;</li>
			<li>Estimate the depth with the anesthesia needle, then again feel it with the narrow scalpel blade and make a 4-5 mm incision through the fascia.&#160; Advance the needle through the incision until it is inserted into the muscle.&#160;</li>
			<li>Collect multiple samples with the window turned in different directions.&#160;Apply continuous suction using a 60 cc syringe while advancing and withdrawing muscle samples into the percutaneous needle two to four times in different directions. Discontinue the suction and remove the needle. 
			NOTE: Each pass (insertion and removal of needle) should take less than a minute.</li>
			<li>Have an assistant apply firm pressure to puncture site for 5 min&#160;to establish hemostasis. Disconnect needle from suction tubing and carefully remove muscle samples from the window and barrel.</li>
		</ol>
		</li>
		<li>Make a second pass if more muscle is needed by repeating the above procedure.</li>
		<li>Remove any visible blood clots from the muscle sample using forceps, weigh the sample, and immediately place in a tube containing ice-cold DPBS. 
		NOTE: The average yield using this methodology is 150 &#177; 20 mg.</li>
	</ol>
	</li>
</ol>
2. Mitochondrial Isolation
<ol>
	<li>Freshly prepare the Chappel-Perry (CP) and Mitochondrial Assay Solution (MAS) buffers (Table 1) on the day of the experiment, or aliquot and store them at -20 &#176;C. Prepare the compounds in dimethyl sulfoxide at a concentration of 2.5 mM and aliquot and store at -20 &#176;C. Use the buffer and compound aliquots stored at -20 &#176;C within 2 months from the day of preparation.</li>
	<li>Remove visible connective tissue from the sample using sharp scissors and tweezers; if needed use a dissecting microscope for this step. Thoroughly wash specimens 3-4 times with ice-cold DPBS buffer to remove blood. Keep the samples on ice-cold DPBS and process as soon as possible, taking care not to exceed more than 45 min&#160;from biopsy. Take precaution to carefully remove any tendons or adipose tissue from the muscle sample.</li>
	<li>Immediately chop the muscle tissue into fine pieces using a sterile pair of scissors and suspend in 500 &#956;l to 1 ml CPI containing proteinase (Nagarse) at a concentration of 0.2 mg/g tissue. Follow this with a 5&#160;min&#160;incubation at RT and then transfer to ice.</li>
	<li>Homogenize the minced tissues treated with Nagarse using an automated homogenizer. Keep the sample on ice throughout this process. Homogenize each tissue sample four times, each time for a pulse of 2 sec, using the automated homogenizer at a speed setting of 10,000 rpm. Wash the probe with 70% ethanol followed by distilled water between tissues.</li>
	<li>Wash the homogenized tissue with an equal volume of CP I (500 &#956;l to 1 ml) and 2x volume of CP II (1 ml to 2 ml), and collect the content in a centrifuge tube and centrifuge at 600 x g, 4 &#176;C for 10 min. Pass the supernatant through a wetted cheese cloth, collect the filtrate and discard the pellet, thus removing the majority of non-mitochondrial fractions.</li>
</ol>
3. Washing the Mitochondria
<ol>
	<li>Centrifuge the supernatant from the above step at 10,000 x g, 4 &#176;C for 10 min. Suspend the pellet in 4 ml of CP II buffer and further centrifuge at 10,000 x g, 4 &#176;C for 10 min. 
	NOTE: On rare occasions, a thin pellet of blood is formed below the mitochondrial pellet. In that case, remove the mitochondrial pellet by gentle aspiration with CPII buffer. This step can be avoided by thoroughly washing away blood at step 2.</li>
	<li>Suspend the pellet obtained in 2ml of CP I buffer. At this stage use a small aliquot from this suspension for protein estimation. Resuspend the remaining sample in CP I buffer and centrifuge as above. Suspend the final pellet in a minimal amount (200 &#956;l) of Mitochondrial Assay Solution (MAS). 
	NOTE: Protein is assayed at this stage because CP I buffer does not have BSA, whereas MAS in which the samples will be later suspended does have BSA in it.</li>
</ol>
4. Estimate the Mitochondrial Content by Measuring Protein Concentration Using a BCA Protein Assay Kit
NOTE: Use this concentration to calculate the quantity of mitochondria used for loading onto a 24-well microplate for respirometric measurements, or for Western blot experiments. Take into account the dilution factor (10) for calculation of the protein concentration.
5. Perform the XF assays as described by Rogers, GW, et al.12
NOTE: Visualize the O2 consumption rate (OCR) in pMoles O2/min, or absolute levels of O2 and pH in the data output.
<ol>
	<li>Use 1x MAS to prepare compounds to be injected. Add a 10x concentration of the compounds to the ports A-D to give a final concentration as follows: Port A, ADP [Adenosine 5&#8217; &#8211;diphosphate, 2 mM, 50 &#956;l]; port B, oligomycin (2 &#956;M, 55 &#956;l); port C, FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, 6 &#181;M, 60 &#956;l]; and port D, 2 &#956;M antimycin-A (65 &#956;l). Prepare sufficient volume of compounds for the required number of wells.</li>
	<li>Determine the optimum amount of mitochondria by titration. For example, load 1.0 &#956;g, 2.5 &#956;g, and 5.0 &#956;g of mitochondria per well in a 50 &#956;l volume of ice-cold 1x MAS containing substrate. To minimize variability between wells, first dilute 10x mitochondria in cold 1x MAS + substrate. Next, deliver 50 &#956;l of this suspension to each well (except background correction wells).</li>
	<li>Centrifuge the plate at 2,000 x g, 20 min&#160;at 4 &#176;C. After centrifugation, gently add 450 &#956;l of 1x MAS + succinate (10 mM) and rotenone (2 &#956;M) (initial conditions; see below) to each well. View the mitochondria under the microscope to ensure homogenous adherence to the well prior to transferring the plate to the XF analyzer. 
	NOTE: The initial conditions used will depend on whether the respiration is driven by either ETC complex I or complex II. For complex II-driven respiration, use succinate (10 mM) and rotenone (2 &#956;M) as initial conditions. Rotenone blocks complex I and succinate provides fuel for complex II (succinate dehydrogenase). To study respiration driven by complex I, use either pyruvate and malate, each at a final concentration of 5 mM or glutamate and malate each at a final concentration of 10 mM as initial conditions. The latter can help in distinguishing dysfunction among tricarboxylic acid cycle and substrate transport. To study respiration driven by both complex I and complex II, include pyruvate and succinate without rotenone or malate as initial conditions. Use palmitoyl carnitine as a substrate for &#946;-oxidation.</li>
	<li>Sequentially measure the mitochondrial respiration in real time using the respirometer by programming it as previously described 12. Use the settings for the respirometer provided in Table 2. 
	NOTE: A brief explanation of various mitochondrial respiration states are as follows:</li>
	<li>State 2 = coupled state with substrate present; State 3 = phosphorylating respiration in the presence of saturating ADP; State 4o = non-phosphorylating respiration induced by oligomycin; State 3u = maximal uncoupled respiration stimulated by the uncoupler FCCP; Residual non-mitochondrial respiration = respiration after complex III inhibition by antimycin-A. It should also be pointed out that, the length of the OCR measurement in combination with the mitochondrial concentration could influence the data by depleting oxygen during the measurement periods.</li>
</ol>
6. Western Blot
NOTE: Determine the mitochondrial marker COX IV and whole tissue GAPDH by Western blotting to assure enrichment of mitochondria in the final sample
<ol>
	<li>Separate the isolated mitochondria and whole tissue extract by 12% sodium dodecyl sulfate polyacrylamide gels.</li>
	<li>Transfer the proteins onto a polyvinylidene fluoride (PVDF) membrane.</li>
	<li>Incubate with COX IV antibody (1:20,000), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. For GAPDH determination, strip the blot and probe with a GAPDH monoclonal antibody (1:2,000). 
	NOTE: If differences in ER contamination are of concern, include ER markers such as ERp72, calnexin, or calreticulin in the analysis.</li>
</ol>

<ol>
	<li>Fernandez-Vizarra, E., 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=Isolation+of+mitochondria+for+biogenetical+studies:+An+update.">Isolation of mitochondria for biogenetical studies: An update.</a> Mitochondrion. 10, 253-262 (2010).</li><li>Fernandez-Vizarra, E., Lopez-Perez, M. J., Enriquez, 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+biogenetically+competent+mitochondria+from+mammalian+tissues+and+cultured+cells.">Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells.</a> Methods. 26, 292-297 (2002).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments : JoVE. , (2011).</li><li>Iglesias-Gonzalez, J., Sanchez-Iglesias, S., Beiras-Iglesias, A., Soto-Otero, R., Mendez-Alvarez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+isolating+rat+brain+mitochondria+with+high+metabolic+activity:+effects+of+EDTA+and+EGTA.">A simple method for isolating rat brain mitochondria with high metabolic activity: effects of EDTA and EGTA.</a> Journal of Neuroscience Methods. 213, 39-42 (2013).</li><li>Gross, V. 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=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Analytical Biochemistry. 418, 213-223 (2011).</li><li>Franko, A., 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=Efficient+isolation+of+pure+and+functional+mitochondria+from+mouse+tissues+using+automated+tissue+disruption+and+enrichment+with+anti-TOM22+magnetic+beads.">Efficient isolation of pure and functional mitochondria from mouse tissues using automated tissue disruption and enrichment with anti-TOM22 magnetic beads.</a> PloS One. 8, e82392 (2013).</li><li>Chappell, J. B., Perry, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+respiratory+and+adenosinetriphosphatase+activities+of+skeletal-muscle+mitochondria.">The respiratory and adenosinetriphosphatase activities of skeletal-muscle mitochondria.</a> The Biochemical Journal. 55, 586-595 (1953).</li><li>Tyrrell, D. J., 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=Respirometric+Profiling+of+Muscle+Mitochondria+and+Blood+Cells+Are+Associated+With+Differences+in+Gait+Speed+Among+Community-Dwelling+Older+Adults.">Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated With Differences in Gait Speed Among Community-Dwelling Older Adults.</a> J Gerontol A Biol Sci Med Sci. , (2014).</li><li>Nicklas, B. J., 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=Relationship+of+physical+function+to+vastus+lateralis+capillary+density+and+metabolic+enzyme+activity+in+elderly+men+and+women.">Relationship of physical function to vastus lateralis capillary density and metabolic enzyme activity in elderly men and women.</a> Aging Clinical and Experimental Research. 20, 302-309 (2008).</li><li>Bergstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+needle+biopsy+of+skeletal+muscle+in+physiological+and+clinical+research.">Percutaneous needle biopsy of skeletal muscle in physiological and clinical research.</a> Scandinavian Journal of Clinical and Laboratory Investigation. 35, 609-616 (1975).</li><li>Rogers, G. W., 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=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PloS One. 6, e21746 (2011).</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, e18317 (2011).</li></ol>

The author, George Rogers, is an employee of Seahorse Bioscience that produces the instrument used in this article. Open Access fees were supported by Seahorse Biosciences.

Isolated mitochondria are often utilized in studies that examine the role of ETC function, as well as other mitochondrial activities, including substrate transport and TCA cycle function. Respirometric assays using isolated organelles permit direct examination of basic processes of oxidative phosphorylation and intrinsic properties of the ETC. Respirometric profiling of isolated mitochondria in comparison to whole cells or permeabilized muscle fibers has the advantages of relatively easy data interpretation and the absence of &#8220;interference&#8221; from non-mitochondrial processes or changes in mitochondrial mass/biogenesis. Normalization of data is based on mitochondrial protein content, thereby allowing straightforward cross-comparison of mitochondria between samples. Respirometric profiling of isolated mitochondria is a preferred approach when the aim of the study is to determine underlying mechanisms and identifying specific targets such as ETC components/complexes, or mitochondrial transport mechanisms.
Described is a protocol for muscle biopsy and isolation of functional mitochondria from small tissue samples. This method yields reproducible results between users due to utilization of an automated homogenizer versus hand operated dounce homogenizers. Isolation of mitochondria can be performed with as little as 20 mg of muscle tissue. The amount of isolated mitochondria that can be obtained from this sample size is sufficient to run Seahorse plate-based respirometry while leaving surplus mitochondria for other experiments and storage for further molecular analyses. It may be noted that this method can be translated to the XF 96, where even smaller amounts of mitochondria can be used (1-2 &#956;g per well).
Several protocols for isolating mitochondria rely on dounce homogenizers for initial tissue disruption. A drawback of this method is the hands-on nature of the initial tissue homogenization. The force and speed of the pestle in the homogenizer can vary significantly between operators 6. This can result in experiment-to-experiment variation, as well as lab-to-lab variation, and leads to difficulty in comparing data between experiments. This is of particular concern in human intervention studies when data from participants are collected at separate time points, typically before and after treatment, and potentially at multiple sites. We use an automated homogenizer for a more consistent approach that yields more reproducible results with limited person-to-person variation. The speed of preparation also makes this approach suitable for handling multiple samples at the same time. Typically, up to three experiments can be performed in a single day.
Potential limitations of the technique described here arise from the use of isolated organelles and the use of a plate-based format. For example, Picard et al. have demonstrated that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. They proposed that mitochondrial isolation techniques result in altered bioenergetic function, such as significantly increased RCR compared to permeabilized myofibers accompanied by greater reactive oxygen species production 13. Compared to permeabilized muscle fibers, isolation of mitochondria does require longer preparation time. Also, loss of cellular content diminishes physiological relevance, something that is retained in whole cells and even permeabilized fibers. The use of plate-based respirometry with the described technique permits replicate runs per sample. However, mitochondria must adhere to the bottom of each well. This configuration is different from their normal environment and may affect functional characteristics. In addition, it should be noted that using this protocol for mitochondrial isolation, there may still be contamination from endoplasmic reticulum (ER) in the mitochondrial preparation. Differences in ER contamination may affect the determination of mitochondrial yield and influence results.
In conclusion, this study presents data that confirms that mitochondria isolated from tissues using this procedure are functionally active and can be used for studies/applications that require high-quality isolated mitochondria from minimal amount of skeletal muscle samples. The advantage of this method is that: i) it is possible to isolate mitochondria from minimal quantities of skeletal muscle, ii) the procedure is quick, iii) with the plate based technology, it is possible to run multiple samples at the same time, and iv) there is enough surplus tissue and isolated mitochondria after the bioenergetic assay for sample storage and other molecular biological investigations.

Mitochondria are the primary energy production sites in the cell and have important roles in aging as well as various age related disorders such as cardiovascular disease, Alzheimer&#8217;s disease, diabetes, cancer, and obesity. Respirometric profiling of isolated mitochondria provides direct analysis of electron transport chain (ETC) function and has contributed significantly to our understanding of mitochondrial biology and its role in health and disease. Isolated mitochondria are used to study various aspects of bioenergetics like, substrate transport, ATP synthase activity, proton leak, etc. The methodology described in this manuscript has been optimized to permit respirometric analysis of mitochondria isolated from skeletal muscle tissue biopsies obtained from human subjects. The biopsy protocol described in this manuscript has been utilized by our staff for the past 12 years. Our group has performed over 700 procedures on adults of various ages, up to 90 years old, and with various chronic disease conditions without any adverse safety issues. A key aspect of this protocol is that it is specifically designed to utilize minimal amounts of tissue, thereby facilitating its use in clinical research studies.
Various protocols have been developed for isolating mitochondria. Fernandez-Vizarra et al.1,2 described a method for isolating mitochondria from various rat tissues as well as cultured cells. Garcia-Cazarin et al.3 have reported a method for isolating mitochondria from skeletal muscles from rat and mouse. A method for isolating mitochondria from rat brain has also been reported by Iglesias-Gonzales et al.4 Gross, et al.5 reported a method of isolating mitochondria using the barocycler and/or the PCT shredder. Recently, Franko et al.6 reported a method of isolating highly enriched mitochondria using anti-TOM22 magnetic beads.
While these protocols yield excellent results, tissue size requirements are high compared to the method described in this manuscript. For example, Gross et al.5 used 1.5-1.8 g of the gastrocnemius muscle, and about 2 g of the kidney tissue. Similarly, Franco et al.6 used 500 mg mouse liver tissue. From our experience, typical yields to be expected from percutaneous needle biopsy of skeletal muscle (Vastus lateralis) range from 100-200 mg. The ability to assess mitochondrial function in 20-50 mg of muscle tissue using the protocol described here permits users to perform multiple assessments per biopsy and to store samples for future use in other molecular biology experiments. This is a critical feature in clinical research and other studies that require diligent use of samples. It should be noted that previously frozen mitochondria are not good for studying coupled respiration due to outer mitochondrial membrane damage and loss of Cytochrome C activity. Our method has been adapted and modified from the method published by Chappell and Perry7.
Using the methods described in this manuscript, we have recently reported that the respirometric profile of mitochondria isolated from human Vastus lateralis is directly correlated with physical ability, measured as gait speed8.

<table><tbody><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Homogenizer Bio-Gen PRO200</td><td>BioExpress</td><td></td><td></td></tr><tr><td>Eppendorf Centrifuge 5804 R</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Deepwell late Rotor&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>6 mm University College Hospital Needle</td><td>Cadence</td><td></td><td></td></tr><tr><td>60 cc syringe</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>96-well plate reader</td><td>Tecan (Genios-basic)</td><td></td><td></td></tr><tr><td>Seahorse XF 24-3 analyzer</td><td>Seahorse Biosciences, Inc.</td><td></td><td></td></tr><tr><td>Protein gel system</td><td>Life Technologies (Invitrogen)</td><td></td><td></td></tr><tr><td>Kodak Gel Logic 112</td><td>Carestream Health, Inc</td><td></td><td></td></tr><tr><td>Kodak camera assembly</td><td>Carestream Health, Inc</td><td></td><td></td></tr><tr><td>Consumables</td><td></td><td></td><td></td></tr><tr><td>XF24 V7 Cell Culture Microplate and XF24 sensor cartridge</td><td>Seahorse Bioscience</td><td>100850-001</td><td></td></tr><tr><td>100867-100</td><td></td><td></td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma-Aldrich Co</td><td>221473</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Acros</td><td>12421-0010</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate buffered saline&amp;nbsp;</td><td>Lonza</td><td>17-512F</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Fisher Scientific</td><td>P333</td><td></td></tr><tr><td>MOPS</td><td>Fisher Scientific</td><td>BP308</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP118</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich Co</td><td>M7506</td><td></td></tr><tr><td>ATP</td><td>Sigma-Aldrich Co</td><td>A9187</td><td></td></tr><tr><td>Fatty acid-free BSA</td><td>Calbiochem</td><td>126575</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich Co</td><td>S0389</td><td></td></tr><tr><td>Bacterial proteinase</td><td>Sigma-Aldrich Co</td><td>P-8038</td><td></td></tr><tr><td>D-Mannitol</td><td>Sigma-Aldrich Co</td><td>M9546</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P284</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich Co</td><td>M9272</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich Co</td><td>H3784</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich Co</td><td>E3889</td><td></td></tr><tr><td>BCA protein assay kit</td><td>Sigma-Aldrich Co</td><td>PI23227</td><td></td></tr><tr><td>Succinic Acid*</td><td>Sigma-Aldrich Co</td><td>S3674</td><td></td></tr><tr><td>Pyruvic acid*</td><td>Sigma-Aldrich Co</td><td>P5280</td><td></td></tr><tr><td>Malic acid*</td><td>Sigma-Aldrich Co</td><td>2288</td><td></td></tr><tr><td>ADP(K&lt;sup&gt;+&lt;/sup&gt; salt)*</td><td>Sigma-Aldrich Co</td><td>A5285</td><td></td></tr><tr><td>XF Cell mito stress test kit</td><td>Seahorse Biosciences</td><td>101706</td><td></td></tr><tr><td>Tween-20</td><td>Santa Cruz Biotechnology, Inc.&amp;nbsp;</td><td>SC-29113</td><td></td></tr><tr><td>NuPAGE 12% Bis-Tris Gel</td><td>Life Technologies (Invitrogen)</td><td>NP0343BOX</td><td></td></tr><tr><td>Immobilin Transfer Membranes (0.45 um)</td><td>Millipore</td><td>IPVH20200</td><td></td></tr><tr><td>MOPS SDS Running Buffer (20X)-500 ml</td><td>Life Technologies (Invitrogen)</td><td>NP0001</td><td></td></tr><tr><td>NuPAGE Transfer Buffer (20X)-1 liter</td><td>Life Technologies (Invitrogen)</td><td>NP0006-1</td><td></td></tr><tr><td>Primary antibodies (mAB to VDAC1/Porin)</td><td>Abcam</td><td>ab14734</td><td></td></tr><tr><td>Primary antibodies (mAB to GAPDH)</td><td>Abcam</td><td>ab9484</td><td></td></tr><tr><td>Anti-Mouse IgG (Goat), HRP-labeled</td><td>PerkinElmer</td><td>NEF822E001EA</td><td></td></tr><tr><td>Anti-Rabbit IgG (Goat), HRP-labeled</td><td>PerkinElmer</td><td>NEF812E001EA</td><td></td></tr><tr><td>*ADP, succinic acid, pyruvic acid, and malic acid should be adjusted to pH 7.4 with KOH only&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

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.

Figure 1 depicts a detailed flowchart of the entire protocol.
Western blot profiles of COX IV/GAPDH (Figure 2) depict the expression of the mitochondrial protein, COX IV, and the non-mitochondrial marker, GAPDH. Expression of both COX IV and GAPDH are evident in the whole muscle lysate. After mitochondria are isolated using the technique described in this protocol, COX IV bands are still evident while GAPDH is absent at the same exposure. Longer exposures may reveal a faint GAPDH band. These blots indicate that the isolated mitochondria have minimal non-mitochondrial contamination. Moreover, COX IV expression in isolated mitochondria is consistent between samples.
Figure 3 shows typical respirometric profiles driven by complex II (succinate and rotenone) using 1.0 &#956;g, 2.5 &#956;g, and 5.0 &#956;g of mitochondria. As expected, overall OCR is increased with higher amounts of mitochondria. The calculated respiratory control ratio (RCR) for this assay is 7.95, indicating that the mitochondrial preparation is of high quality. Furthermore, state 3u OCR is slightly higher than that of state 3, confirming mitochondrial quality.
In order to compare consistency of results when profiling different amounts of mitochondria, we performed ANOVA (analysis of variance) and calculated the sum of squares (SS) as actual values of variance using 1.0 &#181;g, 2.5 &#181;g, and 5.0 &#181;g of mitochondria loaded per well (Figure 4). SS is presented for state 2, state 3, state 3u, antimycin A, and RCR. For state 2 and state 3 measurements, one way ANOVA was statistically significant (p&#60;0.01 and p&#60;0.05, respectively). Similarly, the one way ANOVA was statistically significant for antimycin and RCR (p&#60;0.01 and p&#60;0.0001, respectively. No significant difference was seen for state 3u between groups. These results indicate that 5.0 &#956;g of mitochondria per well gave the lowest SS compared to other concentrations and is the optimal amount to use in the XF 24 system with our population of participants.
Figure 5 serves as a guide to indicate how much mitochondrial protein can be expected based on the initial muscle sample size. As expected there is a strong correlation between the amount of muscle (mg) processed and the total mitochondrial protein content (mg) of the final sample.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="2">Chappel-Perry buffer I (CPI)</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Chappel-Perry buffer II (CPII)</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>0.2 mM</td>
		</tr>
		<tr>
			<td>Fatty-acid free BSA</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Mitochondrial Assay Solution (MAS) (2X)</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>35 mM</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>110 mM</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>2.5 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1.0 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.5 mM</td>
		</tr>
		<tr>
			<td>Fatty-acid free BSA</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Mitochondrial Assay Solution (MAS) with complex II initial conditions</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>1X MAS</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>2 &#956;M</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Mitochondrial Assay Solution (MAS) with complex I initial conditions</td>
		</tr>
		<tr>
			<td>Chemical</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>1X MAS</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>pH</td>
			<td>7.4</td>
		</tr>
		<tr>
			<td colspan="2">*All buffers to be made in deionized water</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Solution and buffer recipes.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Protocol Steps</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">StartProtocol</td>
		</tr>
		<tr>
			<td>Command</td>
			<td>Time (min)</td>
			<td>Port</td>
		</tr>
		<tr>
			<td>Calibrate</td>
			<td>0.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>10.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>0.50</td>
			<td></td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>0.50</td>
			<td></td>
		</tr>
		<tr>
			<td>Inject</td>
			<td></td>
			<td>A</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>6.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Inject</td>
			<td></td>
			<td>B</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Inject</td>
			<td></td>
			<td>C</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Inject</td>
			<td></td>
			<td>D</td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>3.00</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">EndProtocol</td>
		</tr>
	</tbody>
</table>
Table 2. Mix, measure, and mix cycle setting for the respirometer.
<img alt="Figure 1" src="/files/ftp_upload/52350/52350fig1highres.jpg" /> 
Figure 1. Flowchart of the entire protocol. <a href="https://www.jove.com/files/ftp_upload/52350/52350fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52350/52350fig2highres.jpg" /> 
Figure 2. A representative Western blot for whole skeletal muscle tissue as well as isolated mitochondria. Whole tissue extract as well as isolated mitochondria were immunoblotted with COX IV antibody as mitochondrial marker and GAPDH antibody for non-mitochondrial control. No GAPDH band was observed in the isolated mitochondria indicating little or no contamination from non-mitochondrial sources. <a href="https://www.jove.com/files/ftp_upload/52350/52350fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52350/52350fig3highres.jpg" /> 
Figure 3. Representative respirometric profile of mitochondria isolated from human Vastus lateralis. Three concentrations of isolated mitochondria, 5.0 &#956;g, 2.5 &#956;g, and 1.0 &#956;g were used in this assay. Final concentrations of compounds after port injections were 2 mM ADP (port A); 2 &#956;M oligomycin (port B); 6 &#956;M FCCP (port C); and 2 &#956;M antimycin A (port D). Calculated RCR for this run was 7.95. <a href="https://www.jove.com/files/ftp_upload/52350/52350fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52350/52350fig4highres.jpg" /> 
Figure 4. Sum of squares. Sum of squares for the different mitochondrial respiration states and RCR using 5.0 &#956;g, 2.5 &#956;g, and 1.0 &#956;g mitochondria. <a href="https://www.jove.com/files/ftp_upload/52350/52350fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52350/52350fig5highres.jpg" /> 
Figure 5. This can be used as a guide to estimate the amount of mitochondrial protein that can be expected based on the initial muscle sample weight. Regression analysis: of amount of muscle (mg) and total mitochondrial protein yield (mg). As expected, there is a direct positive correlation between the amount of muscle and the total mitochondrial protein obtained.

Watch this Scientific Journal Video about Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy at JoVE.com

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

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 ...

preparation-respirometric-assessment-mitochondria-isolated-from

Research

JoVE Journal

Medicine

19.5K Views.  Wake Forest School of Medicine.  Methoden voor biopsie van vastus lateralis, de voorbereiding van gezuiverde mitochondria, en respirometrische profilering worden beschreven. Het gebruik van kleine spiermassa maakt deze techniek geschikt voor onderzoekstoepassingen. 

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

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

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

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 ...

Biology

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 ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

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 ...

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 ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

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

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