Name: Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy (Video) | JoVE Journal
Uploaded: 2015-02-07T15:00:00.000+00:00
Duration: 11 min 27 s
Description: Learn to isolate mitochondria from skeletal muscle via percutaneous needle biopsy for respirometric assessment. This minimal-tissue technique enables clinical study of mitochondrial respiratory function.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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/pubmed?term=((Isolation+of+mitochondria+for+biogenetical+studies%3A+An+update%5BTitle%5D)+AND+%22Mitochondrion%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+biogenetically+competent+mitochondria+from+mammalian+tissues+and+cultured+cells%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+isolation+from+skeletal+muscle%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+%3A+JoVE%22%5BJournal%5D)">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/pubmed?term=((A+simple+method+for+isolating+rat+brain+mitochondria+with+high+metabolic+activity%3A+effects+of+EDTA+and+EGTA%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">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/pubmed?term=((Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Efficient+isolation+of+pure+and+functional+mitochondria+from+mouse+tissues+using+automated+tissue+disruption+and+enrichment+with+anti-TOM22+magnetic+beads%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((The+respiratory+and+adenosinetriphosphatase+activities+of+skeletal-muscle+mitochondria%5BTitle%5D)+AND+%22The+Biochemical+Journal%22%5BJournal%5D)">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/pubmed?term=((Respirometric+Profiling+of+Muscle+Mitochondria+and+Blood+Cells+Are+Associated+With+Differences+in+Gait+Speed+Among+Community-Dwelling+Older+Adults%5BTitle%5D)+AND+%22J+Gerontol+A+Biol+Sci+Med+Sci%22%5BJournal%5D)">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/pubmed?term=((Relationship+of+physical+function+to+vastus+lateralis+capillary+density+and+metabolic+enzyme+activity+in+elderly+men+and+women%5BTitle%5D)+AND+%22Aging+Clinical+and+Experimental+Research%22%5BJournal%5D)">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/pubmed?term=((Percutaneous+needle+biopsy+of+skeletal+muscle+in+physiological+and+clinical+research%5BTitle%5D)+AND+%22Scandinavian+Journal+of+Clinical+and+Laboratory+Investigation%22%5BJournal%5D)">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/pubmed?term=((High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+structure+and+function+are+disrupted+by+standard+isolation+methods%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">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

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 μg, 2.5 μg, and 5.0 μg 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 µg, 2.5 µg, and 5.0 µg 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&lt;0.01 and p&lt;0.05, respectively). Similarly, the one way ANOVA was statistically significant for antimycin and RCR (p&lt;0.01 and p&lt;0.0001, respectively. No significant difference was seen for state 3u between groups. These results indicate that 5.0 μg 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 μM</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. 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="Mitochondria preparation steps flowchart; includes biopsy, protein assay, centrifugation process." src="/files/ftp_upload/52350/52350fig1highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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="Western blot of COX IV and GAPDH in raw lysate vs purified mitochondria; protein comparison." src="/files/ftp_upload/52350/52350fig2highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
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="Mitochondrial respiration graph; OCR vs. time, ADP, oligomycin, FCCP, antimycin-A analysis." src="/files/ftp_upload/52350/52350fig3highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
Figure 3. Representative respirometric profile of mitochondria isolated from human Vastus lateralis. Three concentrations of isolated mitochondria, 5.0 μg, 2.5 μg, and 1.0 μg were used in this assay. Final concentrations of compounds after port injections were 2 mM ADP (port A); 2 μM oligomycin (port B); 6 μM FCCP (port C); and 2 μM 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="Mitochondrial respiration bar charts; basal, State 3/3u, antimycin A impact, statistical P-values." src="/files/ftp_upload/52350/52350fig4highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
Figure 4. Sum of squares. Sum of squares for the different mitochondrial respiration states and RCR using 5.0 μg, 2.5 μg, and 1.0 μg 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="Muscle and protein correlation analysis; scatter plot shows linear regression, y=mx+c, slope=0.0062." src="/files/ftp_upload/52350/52350fig5highres.jpg" data-altupdatedat="1787630408000" data-alt="Figure 5"> 
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.

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 ...

The overall goal of this procedure is to examine respiratory control in mitochondria, isolated from biopsied skeletal muscle tissue. This is accomplished by first obtaining a small amount of muscle by percutaneous needle biopsy. The second step is to isolate mitochondria from the biopsied muscle by homogenization.Next, the isolated mitochondria are washed and separated from the non mitochondrial fractions through centrifugation and passing the sample through a wedded cheesecloth. The final step is to plate isolated mitochondria to perform respiro metric analysis through extracellular flux assays to visualize the oxygen consumption rate. Ultimately, the seahorse extracellular flux analyzer is used to examine respiratory control in isolated mitochondria.The main advantage of this technique over existing methods is that it requires a minimum amount of muscle tissue as low as 20 milligrams and limits inter experimental variability. These features make this technique suitable for clinical research applications. The implications for this technique are for diagnosing mitochondrial dysfunction in patients.Visual demonstration of this method is critical as the muscle biopsy and mitochondrial isolation require precise manipulations that are best demonstrated visually rather than solely described in text. To perform the biopsy locally, administer 1%lidocaine, taking care not to infiltrate the muscle. Follow this with a 10 minute wait period.To permit sufficient numbing, take the biopsies from the middle region of muscle between insertion and origin, avoiding subfascial and myo tendonous areas. To achieve this first, use a percutaneous needle and follow a fascial pop or resistance of the fascia as a guide. Estimate the depth with the anesthesia needle, then feel it with the narrow scalpel blade and make a four to five millimeter incision through the fascia.Advance the needle through the incision until it is inserted into the muscle. Collect multiple samples with the window turned in different directions. Apply continuous suction using a 60 cubic centimeter 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. Have an assistant apply firm pressure to the puncture site for five minutes. To establish hemostasis, disconnect the needle from suction tubing and carefully remove the muscle samples from the window and barrel.Make a second pass if more muscle is needed by repeating this procedure. Remove any visible blood clots from the muscle sample using forceps. Then weigh the sample and immediately place in a tube containing ice.Cold ECCOs phosphate buffered saline or DPBS. Remove visible connective tissue from the sample using sharp scissors and tweezers.Thoroughly. Wash specimens three to four times with ice cold DPBS buffer.To remove blood, keep the samples on ice cold DPBS and process as soon as possible. Within 45 minutes of the biopsy, take precaution to carefully remove any tendons or adipose tissue from the muscle sample. Remove a small portion of any extra tissue for storage at negative 80 degrees Celsius.This may be used for other experiments such as Western blots. Immediately chop the muscle tissue into fine pieces. Using a sterile pair of scissors, then suspend in 500 microliters to one milliliter of a Sistine protein ACE inhibitor, such as nagars dissolved in CP one buffer at a concentration of 0.2 milligrams per gram of tissue.Mince the sample until it appears well suspended. Follow this with a five minute incubation at room temperature before transferring to ice, making sure that the homogenizer is thoroughly washed.Beforehand. Homogenize the minced tissue treated with nagars.Using an automated homogenizer. Keep the sample on ice throughout this process. Homogenize each tissue sample four times each time for a pulse of two seconds.Using the automated homogenizer at a speed setting of 10, 000 RPM, place the sample back on ice. Wash the probe with 70%ethanol followed by distilled water between tissues. Now, wash the homogenized tissue with an equal volume of chapel Perry one or CP one buffer.Then wash the tissue with twice the volume of CP two buffer. Add the balance tube by bringing water up to the same volume in another conical tube. Collect the content in a centrifuge tube and centrifuge at 600 GS four degrees Celsius or 10 minutes pre-wet the cheesecloth to allow all particles to pass through and not be caught on the cloth.Pass the supernatant through a wedded cheesecloth, collecting the filtrate and discarding the pellet, thus removing the majority of non mitochondrial fractions. Then ultracentrifuge the supernatant at 10, 000 GS four degrees Celsius for 10 minutes. Suspend the pellet in four milliliters of CP two buffer before centrifuging a second time in the same manner following centrifugation.Discard the supernatant and resuspend the pate in two milliliters of CP one buffer. At this point, place a small aliquot from this suspension into a separate test tube to be used for protein estimation. Resus, suspend the remaining sample in CP one buffer and centrifuge.As before, note that the protein plate should be set up with the standards during earlier centrifugation steps. Finally, resuspend the final pellet in a minimal amount of mitochondrial assay solution or mass. Perform XF assays to visualize the oxygen consumption rate or OCR in pico moles of oxygen per minute or absolute levels of oxygen and pH.In the data output. Add a 10 x concentration of the compounds in one X mass to the ports A through D of the plate based respirometer to give a final concentration as listed in the text protocol. Prepare sufficient volume of compounds for the required number of wells.Use the protein calculations measured earlier to determine the concentrations of mitochondria to be added. Determine the optimum amount of mitochondria by titrating one microgram, 2.5 micrograms and five micrograms of mitochondria per well. Of the 24 well plate to minimize variability between wells.First dilute 10 x mitochondria in cold, one x mass plus substrate. Next, deliver 50 microliters of this suspension to each well. Leaving four wells containing only 50 microliters of mass to be used as background readings.Place the pre soaked calibration plate along with the upper cartridge from earlier into the respirometer to perform the instrument calibration. Preparing for the test plate run centrifuge the plate at 2000 GS for 20 minutes at four degrees Celsius. After centrifugation.Gently add 450 microliters of one x mass, plus succinate and rotten known 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.Sequentially. Measure the mitochondrial respiration in real time using the respirometer by programming it using the settings for the respirometer provided in the text protocol shown here are typical Respiro metric profiles driven by complex two using one microgram, 2.5 micrograms, and five micrograms of mitochondria as expected.Overall, OCR is increased with higher amounts of mitochondria. The calculated respiratory control ratio or RCR for this assay is 7.95, indicating that the mitochondrial preparation is of high quality in order to compare consistency of results when profiling different amounts of mitochondria analysis of variants or Inova was performed and the sum of squares was calculated. Sum of squares or SS is presented for state two, state three, state three, uncoupled antimycin A and RCR for state two and state three measurements one way.Innova was statistically significant as well as for ANTIMYCIN and RCR. No significant difference was seen for state three uncoupled between groups. These results indicate that five micrograms of mitochondria per well gave the lowest SS compared to other concentrations.This plot 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 processed and the total mitochondrial protein content of the final sample. Once mastered, this technique can be completed in less than three hours if it is performed properly.Following the muscle biopsy, it is important to make sure that the patient does not take any aspirin or ibuprofen or other over-the-counter non-steroidals for the next three days in order to ensure that there is good healing of the tissues that we have biopsied. The next thing is that we wanna make sure that they don't do any strenuous activity also for the next day and a half, so nothing like jarring. That would be basketball trampoline tillery, but they can do their own normal activities.We also ask that they keep the area dry for 24 hours, but the first time that it gets wet to be in a shower so that any types of bacteria that may be on the skin that we have not cleaned does not get into the incision. After watching this video, you should have a better understanding of how to biopsy S lateral skeletal muscle tissue, isolate mitochondria, and measure respiratory control.

preparation-respirometric-assessment-mitochondria-isolated-from

Research

JoVE Journal

Medicine

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.

Mitochondrial Isolation from Skeletal Muscle

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 &lt;30 min compared to 60 - 100 min using alternative methods.

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

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.

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

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.

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

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.

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

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

Mitochondria are key metabolic organelles that exhibit a high level of phenotypic plasticity in skeletal muscle. The import of proteins from the cytosol is a critical pathway for organelle biogenesis, essential for the expansion of the reticulum and the maintenance of mitochondrial function.  Therefore, protein import serves as a barometer of cellular health.

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria

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.

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

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.

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

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.

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

Learn to isolate mitochondria from skeletal muscle via percutaneous needle biopsy for respirometric assessment. This minimal-tissue technique enables clinical study of mitochondrial respiratory function.

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy (Video) | JoVE Journal

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

Explore More Videos