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 border="0" cellpadding="0" cellspacing="0" width="593">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Equipment</td>
			<td style="width:201px;"></td>
			<td style="width:115px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of the Equipment</td>
			<td>Company</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Homogenizer Bio-Gen PRO200</td>
			<td>BioExpress</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Centrifuge 5804 R</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deepwell late Rotor&#160;</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6mm University College Hospital Needle</td>
			<td>Cadence</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60cc syringe</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well plate reader</td>
			<td>Tecan (Genios-basic)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seahorse XF 24-3 analyzer</td>
			<td>Seahorse Biosciences, Inc.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein gel system</td>
			<td>Life Technologies (Invitrogen)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kodak Gel Logic 112</td>
			<td>Carestream Health, Inc</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kodak camera assembly</td>
			<td>Carestream Health, Inc</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Consumables</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;">XF24 V7 Cell Culture Microplate and XF24 sensor cartridge</td>
			<td rowspan="2">Seahorse Bioscience</td>
			<td>100850-001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100867-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich Co</td>
			<td>221473</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid (HCl)</td>
			<td>Acros</td>
			<td>12421-0010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate buffered saline&#160;</td>
			<td>Lonza</td>
			<td>17-512F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P333</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MOPS</td>
			<td>Fisher Scientific</td>
			<td>BP308</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td>BP118</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Magnesium sulfate (MgSO4)</td>
			<td>Sigma-Aldrich Co</td>
			<td>M7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP</td>
			<td>Sigma-Aldrich Co</td>
			<td>A9187</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fatty acid-free BSA</td>
			<td>Calbiochem</td>
			<td>126575</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma-Aldrich Co</td>
			<td>S0389</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacterial proteinase</td>
			<td>Sigma-Aldrich Co</td>
			<td>P-8038</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Mannitol</td>
			<td>Sigma-Aldrich Co</td>
			<td>M9546</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P284</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich Co</td>
			<td>M9272</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich Co</td>
			<td>H3784</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma-Aldrich Co</td>
			<td>E3889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCA protein assay kit</td>
			<td>Sigma-Aldrich Co</td>
			<td>PI23227</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid*</td>
			<td>Sigma-Aldrich Co</td>
			<td>S3674</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid*</td>
			<td>Sigma-Aldrich Co</td>
			<td>P5280</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Malic acid*</td>
			<td>Sigma-Aldrich Co</td>
			<td>2288</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ADP(K+ salt)*</td>
			<td>Sigma-Aldrich Co</td>
			<td>A5285</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XF Cell mito stress test kit</td>
			<td>Seahorse Biosciences</td>
			<td>101706</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Santa Cruz Biotechnology, Inc.&#160;</td>
			<td>SC-29113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE 12% Bis-Tris Gel</td>
			<td>Life Technologies (Invitrogen)</td>
			<td>NP0343BOX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilin Transfer Membranes (0.45um)</td>
			<td>Millipore</td>
			<td>IPVH20200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MOPS SDS Running Buffer (20X)-500 ml</td>
			<td>Life Technologies (Invitrogen)</td>
			<td>NP0001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuPAGE Transfer Buffer (20X)-1 liter</td>
			<td>Life Technologies (Invitrogen)</td>
			<td>NP0006-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies (mAB to VDAC1/Porin)</td>
			<td>Abcam</td>
			<td>ab14734</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies (mAB to GAPDH)</td>
			<td>Abcam</td>
			<td>ab9484</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG (Goat), HRP-labeled</td>
			<td>PerkinElmer</td>
			<td>NEF822E001EA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Rabbit IgG (Goat), HRP-labeled</td>
			<td>PerkinElmer</td>
			<td>NEF812E001EA</td>
		</tr>
		<tr height="20">
			<td colspan="3" height="20" style="height:20px;">*ADP, succinic acid, pyruvic acid, and malic acid should be adjusted to pH 7.4 with KOH only&#160;</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

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JoVE Journal

Medicine

19.1K vues.  Wake Forest School of Medicine.  Méthodes pour la biopsie du vaste externe, préparation de mitochondries purifiées, et le profilage respirométrique sont décrits. L'utilisation de petit volume musculaire rend cette technique convient pour des applications de recherche clinique. 

Vidéo: 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 ...

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 ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.

Substernal thyroid lesions are common, and need to be differentiated from malignancy. Obtaining percutaneous fine needle biopsy is not possible due to its retrosternal location. This article proposes a protocol for biopsy of substernal thyroid lesions using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA).

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real time images with a 15 &#181;m axial resolution at an effective tissue penetration of 2-3 mm. Within the OCT images the loss of signal intensity per millimeter of tissue penetration, the attenuation coefficient, is calculated. The attenuation coefficient is a tissue specific property, providing a quantitative parameter for tissue differentiation. 
Until now, renal mass treatment decisions have been made primarily on the basis of MRI and CT imaging characteristics, age and comorbidity. However these parameters and diagnostic methods lack the finesse to truly detect the malignant potential of a renal mass. A successful core biopsy or fine needle aspiration provides objective tumor differentiation with both sensitivity and specificity in the range of 95-100%. However, a non-diagnostic rate of 10-20% overall, and even up to 30% in SRMs, is to be expected, delaying the diagnostic process due to the frequent necessity for additional biopsy procedures. 
We aim to develop OCT into an optical biopsy, providing real-time imaging combined with on-the-spot tumor differentiation. This publication provides a detailed step-by-step approach for percutaneous, needle based, OCT of renal masses.

In Vivo, Percutaneous, Needle Based, Optical Coherence Tomography of Renal Masses

Optical coherence tomography (OCT) is a high resolution imaging technique that allows analysis of tissue specific optical properties providing the means for tissue differentiation. We developed needle based OCT, providing real-time imaging combined with on-the-spot tumor differentiation. This publication describes a method for percutaneous, needle based OCT of renal masses.

Optical coherence tomography (OCT) is the optical equivalent of ultrasound imaging, based on the backscattering of near infrared light. OCT provides real ...

Povidone Iodine Rectal Preparation at Time of Prostate Needle Biopsy is a Simple and Reproducible Means to Reduce Risk of Procedural Infection

Single institution and population-based studies highlight that infectious complications following transrectal ultrasound guided prostate needle biopsy (TRUS PNB) are increasing. Such infections are largely attributable to quinolone resistant microorganisms which colonize the rectal vault and are translocated into the bloodstream during the biopsy procedure. A povidone iodine rectal preparation (PIRP) at time of biopsy is a simple, reproducible method to reduce rectal microorganism colony counts and therefore resultant infections following TRUS PNB.
All patients are administered three days of oral antibiotic therapy prior to biopsy. The PIRP technique involves initially positioning the patient in the standard manner for a TRUS PNB. Following digital rectal examination, 15 ml&#160;of a 10% solution of commercially available povidone iodine is mixed with 5 ml&#160;of 1% lidocaine jelly to create slurry. A 4 cm x 4 cm sterile gauze is soaked in this slurry and then inserted into the rectal vault for 2 min after which it is removed. Thereafter, a disposable cotton gynecologic swab is used to paint both the perianal area and the rectal vault to a distance of 3 cm from the anus. The povidone iodine solution is then allowed to dry for 2 - 3 min prior to proceeding with standard transrectal ultrasonography and subsequent biopsy.
This PIRP technique has been in practice at our institution since March of 2012 with an associated reduction of post-biopsy infections from 4.3% to 0.6% (p = 0.02). The principal advantage of this prophylaxis regimen is its simplicity and reproducibility with use of an easily available, inexpensive agent to reduce infections. Furthermore, the technique avoids exposing patients to additional systemic antibiotics with potential further propagation of multi-drug resistant organisms. Usage of PIRP at TRUS PNB, however, is not applicable for patients with iodine or shellfish allergies.

Here, we present a protocol for use at the time of transrectal ultrasound guided prostate needle biopsy (TRUS PNB) that is a simple and cost-effective means to reduce infections following the procedure.

Single institution and population-based studies highlight that infectious complications following transrectal ultrasound guided prostate needle biopsy ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...