The Fralin Life Science Research Institute and The Metabolic Phenotyping Core at Virginia Tech supported this work.

NOTE: This protocol begins after one has isolated mitochondria from red skeletal muscle. Mitochondria were isolated as previously described from ~ 75 - 100 mg of red skeletal muscle13. Between 5 - 10 &#181;g/&#181;l of mitochondrial protein will be attained from this amount of tissue by resuspending the final mitochondrial pellet in &#8804;50 &#181;l of isolation buffer for mitochondria (IBM) 2 (See Frisard et al13).
1. Setup
<ol>
	<li>Prepare working stock solutions (Table 1) and mitochondrial assay solution (MAS) mixes (Table 2) for subsequent assays. 
	Note: Most of the preparation for the assays can be done in advance as most stocks solutions and MAS can be stored.</li>
	<li>Hydrate a respirometric assay cartridge in 1 ml calibration solution the night before the experiment in a non-CO2 incubator at 37 &#176;C.</li>
	<li>The day of the experiment, take out frozen stocks (substrates and mitochondrial modulators, MAS mix, MAS w/o BSA, Table 1) and thaw in 37 &#176;C bath or incubator.</li>
	<li>Prepare all substrate solutions (Table 3) and injections (Table 4-5) and adjust pH as desired (pH&#160;7.4). After the pH is adjusted, store on ice.</li>
	<li>Program the multi-well oxygen consumption measurement machine to proper mix, wait, and measure parameters (See Table 6) and label background and group/condition wells by first entering the analysis software, followed by the &#8220;Standard&#8221; mode.
	<ol>
		<li>Enter into the &#8220;Guest&#8221; forum and click on the &#8220;Assay Wizard&#8221;. Once in the &#8220;Assay Wizard&#8221;, click on &#8220;Protocol&#8221; to change mix, wait, and measure parameters. Label the background wells under the &#8220;Back. Correction&#8221; tab and be sure to highlight &#8220;Do background correction&#8221;.</li>
		<li>Label conditions by first clicking on the &#8220;Groups &#38; Labels&#8221; tab, followed by the &#8220;Group Info&#8221; tab. After these parameters are entered, click &#8220;End&#8221;, followed by &#8220;Save Template&#8221;.</li>
	</ol>
	</li>
</ol>
2. The Assay Run
<ol>
	<li>Load the injection solutions into an assay run cartridge and start the calibration by first entering the analysis software, followed by entering the &#8220;Standard&#8221; mode. Enter into the &#8220;Guest&#8221; forum. Open the template saved in Step 1.5 under the &#8220;XF&#8221; drive, under the &#8220;Templates&#8221; tab. Once open, click &#8220;Start&#8221; to begin calibration. 
	Note: The assay run cartridge must be entered into the machine with the notched corner to the bottom left. This takes about 30 min. Take care to inject solutions into the proper ports. Injections in Table 4-5 are listed in order of loading.</li>
	<li>Gently, but thoroughly mix the mitochondria stock by stirring the solution with a 200 &#181;l pipette tip, followed by gently triturating the stock with a 200 &#181;l pipette tip that has had its orifice widened by cutting ~ 3 mm off the end of the tip with scissors.</li>
	<li>Perform a bicinchoninic acid (BCA) assay to determine the protein concentration of the mitochondrial stock.</li>
	<li>Using the stock concentration acquired from step 2.3, resuspend 10&#160;&#181;g of mitochondrial protein/200&#160;&#181;l of the succinate/ rotenone substrate mix (Table 3). Resuspend 14 &#181;g of mitochondrial protein/ 200 &#181;l of the remaining substrate mixes (This must be done for each substrate mix) (Table 3). Place all mitochondrial protein/substrate mixes on ice. 
	Note: The optimal amount of mitochondrial protein per well for each assay was determined as previously described9. It was determined that the pyruvate /malate, palmitoyl carnitine/malate, glutamate/malate and electron flow assays utilizes 3.5 &#181;g of mitochondrial protein, while the succinate/rotenone assay utilizes 2.5 &#181;g of mitochondrial protein per well. Therefore, the researcher needs to resuspend enough mitochondrial protein for 4 replicates in this step since either 2.5 &#181;g or 3.5 &#181;g per 50 &#181;l is loaded per well (See step 2.6).</li>
	<li>Mix the mitochondria/ substrate solutions gently, but thoroughly by stirring the solution with a 200 &#181;l pipette tip, followed by gently triturating the stock with a 200 &#181;l pipette tip that has had ~ 3 mm of its end cut off.</li>
	<li>Place a cell culture plate on ice and in triplicate, load 50 &#181;l/well of each of the mitochondria/substrate mixes. The developers of the microplate based respirometric assay recommend leaving a minimum of two blank (no mitochondria) wells, preferably on either side on the cell culture plate.</li>
	<li>Spin the cell culture plate at 2,000 g for 20 min at 4 &#176;C. While the plate is spinning, warm up the substrate solutions in a 37 &#176;C water bath.</li>
	<li>After the spin is complete, carefully load 450 &#181;l of each substrate solution on top of its respective wells (i.e., load the pyruvate/malate substrate to the wells that have mitochondria initially resuspended in this substrate solution). Be sure to load the plate at RT. Note: Blank wells should be loaded with 500 &#181;l of substrate. Blank wells designated for the electron flow assay should be loaded with the electron flow substrate (Pyruvate/Malate + FCCP), while the coupled assay blank wells can be loaded with any coupling assay substrate. 
	Note: If performing the coupled and electron flow assays on the same plate, the researcher must reassign background wells to the respective assays after the assay run is complete using the &#8220;XF Reader&#8221; platform via the &#8220;Instrument&#8221; tab. Once within the &#8220;Instrument&#8221; setting, click on the &#8220;Administration&#8221; tab, followed by &#8220;Background Correction&#8221;. Hit &#8220;End Instrument Setup Mode&#8221; when complete. This is because the combination of ascorbate/TMPD can consume O2, thus a separate background correction is needed for each assay type.</li>
	<li>After the 450 &#181;l of substrate is added to each well, view the layer of mitochondria in the well to ensure the mitochondria are dispersed evenly in a single monolayer (20X magnification [See Rogers9, Figure 4]). Wells that do not appear to have an evenly distributed monolayer can be removed post hoc.</li>
	<li>After checking for mitochondrial adherence, insert the microplate into the respirometric assay instrument and start the assay run by clicking &#8220;OK&#8221;. 
	Note: The calibration from step 2.1 must be complete before the run will begin. Once the researcher clicks&#160;&#8220;OK&#8221;, the machine will eject the utility plate that was used for calibration.
	<ol>
		<li>Remove the utility plate and place the cell culture plate that contains the mitochondria on the tray. The blue notch on the cell culture plate should be placed in the bottom left corner of the tray. Click on, &#8220;continue&#8221; to start the assay run.</li>
	</ol>
	</li>
	<li>After the run is complete, eject the cell culture plate and cartridge by pressing &#8220;Eject&#8221; on the screen.
	<ol>
		<li>Once the plate is ejected, remove and discard the cell culture plate and cartridge and click &#8220;Continue&#8221; on the screen. The run will be automatically saved as an .xls file in the &#8220;Data&#8221; file.</li>
	</ol>
	</li>
	<li>Open this file and change the display from &#8220;O2&#8221; to &#8220;OCR&#8221; by hitting the downward arrow under the &#8220;Y1:&#8221; marker.
	<ol>
		<li>Next change &#8220;Middle Point&#8221; to &#8220;Point-to-Point Rates&#8221; under the &#8220;Rate Data Displayed As:&#8221; option. Click &#8220;OK&#8221; to apply these changes.</li>
	</ol>
	</li>
	<li>Click the &#8220;Well Group Mode&#8221; tab on bottom left of the screen to group wells of similar conditions together. Finally, click the &#8220;Sample Mean/Standard Error&#8221; tab toward the middle of the screen, to the left. Alternatively, these commands can be setup before the assay begins in the &#8220;Assay Wizard&#8221; setting. 
	Note: Each condition will have a mean &#177; standard deviation displayed for each rate and each condition. There are rate measurements taken per condition&#160;(State 2[Coupling]/State3u [Electron Flow] respiration followed by four injections). Use the mean of the State 2 rate of the second measurement, determine State 4o at the minimum point after oligomycin injection, use the maximum point for State 3 and State 3u after ADP and FCCP injection, respectively, and use the minimum point for Antimycin A induced respiration for the Coupling Assays. Use the maximum point for pyruvate/malate induced State 3u respiration, use the minimum point for rotenone induced respiration, use the maximum point for succinate induced State 3u respiration, and use the minimum point for Antimycin A induced respiration for the Electron Flow assay. All experiments were performed 3 times and the data shown are the results of a representative&#160;tracing.</li>
</ol>

<ol>
	<li>Brand, M., Nicholls, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochem J. 435, 297-312 (2011).</li><li>Wang, H., Hiatt, W. R., Barstow, T. J., Brass, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+muscle+mitochondrial+DNA+content,+mitochondrial+enzyme+activity+and+oxidative+capacity+in+man:+alterations+with+disease.">Relationships between muscle mitochondrial DNA content, mitochondrial enzyme activity and oxidative capacity in man: alterations with disease.</a> European Journal of Applied Physiology and Occupational Physiology. 80, 22-27 (1999).</li><li>Lauri, A., Pompilio, G., Capogrossi, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+genome+in+aging+and+senescence.">The mitochondrial genome in aging and senescence.</a> Ageing Research Reviews. 18C, 1-15 (2014).</li><li>Nunnari, J., Suomalainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+in+sickness+and+in+health.">Mitochondria: in sickness and in health.</a> Cell. 148, 1145-1159 (2012).</li><li>Dube, J. 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=Effects+of+acute+lipid+overload+on+skeletal+muscle+insulin+resistance,+metabolic+flexibility,+and+mitochondrial+performance.">Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance.</a> Endocrinology and Metabolism. 307, E1117-E1124 (2014).</li><li>Disease Control and Prevention Services. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+diabetes+statistics+report:+estimates+of+diabetes+and+its+burden+in+the+United+States.">National diabetes statistics report: estimates of diabetes and its burden in the United States.</a> , (2014).</li><li>Guarino, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+determining+oxygen+consumption+rates+of+static+cultures+from+microplate+measurements+of+pericellular+dissolved+oxygen+concentration.">Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration.</a> Biotechnology and Bioengineering. 86, 775-787 (2004).</li><li>Will, Y., Hynes, J., Ogurtsov, V. I., Papkovsky, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+function+using+phosphorescent+oxygen-sensitive+probes.">Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes.</a> Nature Protocols. 1, 2563-2572 (2007).</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>Wu, 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=Multiparameter+metabolic+analysis+reveals+a+close+link+between+attenuated+mitochondrial+bioenergetic+function+and+enhanced+glycolysis+dependency+in+human+tumor+cells.">Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.</a> American Journal of Physiology-Cell Physiology. 292, C125-C136 (2007).</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 (San Diego, Calif.). 26, 292-297 (2002).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nature Protocols. 2, 287-295 (2007).</li><li>Frisard, M. I., 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=Low+levels+of+lipopolysaccharide+modulate+mitochondrial+oxygen+consumption+in+skeletal+muscle.">Low levels of lipopolysaccharide modulate mitochondrial oxygen consumption in skeletal muscle.</a> Metabolism. 64, 416-427 (2015).</li><li>Chance, B., Williams, G. R. <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+chain+and+oxidative+phosphorylation.">The respiratory chain and oxidative phosphorylation.</a> Advances in Enzymology and Related Subjects of Biochemistry. 17, 65-134 (1956).</li><li>Gnaiger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathways+and+respiratory+control.">Mitochondrial pathways and respiratory control.</a> , (2007).</li></ol>

George W. Rogers is an employee of Seahorse Bioscience that manufactures the instrument for which these assays were developed. Open Access fees were provided by, Seahorse Bioscience, a part of Agilent Technologies.

The methods presented in this article provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using mitochondria isolated from 75 - 100 mg of mouse skeletal muscle. These assays can be performed with high precision as evidenced by the tight standard deviation between triplicate wells. Importantly, these respirometric assays allow pinpointing of where abnormalities and/or adaptations may be occurring in the ETC, TCA cycle, &#946;-oxidation pathway, substrate transporters, etc. in a commonly used animal model.
It is important to highlight the rationale for using various fuels and inhibitors used in this protocol. The pyruvate/malate and glutamate/malate respirometric assays allow for the assessment of Complex I mediated respiration, as well as the assessment of their respective transporters, and in the case of glutamate, the deaminase15. Alternatively, the combination of succinate/rotenone allows the assessment of mitochondrial respiratory flux through Complex II of the ETC since rotenone inhibits complex I and succinate provides electrons to Complex II via the reduction of flavin adenine dinucleotide (FADH2)15. These assays provide substrate specific information as to coupling efficiency and maximal respiration. The electron flow assay is unique in that the combination of substrates and inhibitors allows for the assessment of multiple complexes during mitochondrial respiratory flux9. The initial substrate mix of pyruvate/malate + FCCP allows for the evaluation of maximal respiration driven by Complex I, while the injection of rotenone followed by succinate allows for the assessment maximal respiration driven by Complex II. The injection of Antimycin A, an inhibitor of Complex III, followed by the injection of ascorbate/TMPD allow the for the evaluation of respiration driven by Complex IV since the Ascorbate/TMPD is an electron donor to Cytochrome C/Complex IV. While no information on coupling efficiency is obtained, the method is ideal for very small sample sizes that preclude running multiple substrates independently. Finally, the use of palmitoyl carnitine/malate allows for the assessment of the coordination between &#946;-oxidation and the ETC since the reducing equivalent produced from the oxidation of palmitic acid (&#946;-oxidation) feed into the ETC through the electron transferring flavoprotein15. It should be noted that the Coupling and Electron Flow assays could also be used in tandem to identify changes in mitochondrial function due to some intervention (drug treatment, genetic manipulation).
The high precision attained for these assays is primarily due to thorough mixing of the mitochondria, whether it is prior to protein determination, or with the substrate solutions. Along these lines, once the mitochondria is resuspendend in the substrate solutions, it is critical to mix this solution thoroughly prior to loading the cell culture plate as described in Step 2.2 with a widened orifice pipette tip. Failure to mix the mitochondria thoroughly will lead to large variation within the assay. In addition, using a narrow orifice pipette tip will create shearing forces while mixing the mitochondria and increases the potential to damage the mitochondrial membranes and release of Cytochrome C. The adherence step (2.7) is also a critical step in this protocol. Failure to spin the loaded cell culture plate long/fast enough will result in incomplete adherence of the mitochondria to the well, thus leading increased variability between wells and measurements.
The described protocol has been optimized to include: loading an optimal amount of mitochondrial protein per well, using the correct concentrations/preparation methods to make stock and substrate solutions, altering the assay run to ensure mitochondrial state plateaus, and appropriate mixing of the mitochondrial stock and mitochondrial/substrate mixes. Prior to these optimization efforts, the authors encountered pitfalls in the assay run. The following discusses troubleshooting methods/modifications that were helpful in optimizing this protocol. With respect to optimal loading, loading too little mitochondria will not elicit a robust response, while loading too much mitochondria will exhaust the oxygen within the microchamber and lead to inaccurate measurements. Rogers et al9 provides&#160;examples of determining optimal loading amounts of mitochondria per well for microplate based respirometric assays. More often, too much mitochondria is loaded per well as evidenced by state 2 rates over 100-200 pmol/min/well and state 3 rates &#62;1500 pmol/min/well. If over loading occurs, perform an experiment with varying concentration of mitochondrial protein (e.g., between 1 - 10 &#956;g) to elicit OCRs within the dynamic range of the oxygen consumption measurement machine. Preparing and using the correct concentrations of substrates and stocks is of utmost importance. Always use the acid form of substrates/injections and adjust the pH with potassium hydroxide or HCl; sodium buffers/solutions are not recommended. In addition, resuspending substrates/stocks in DMSO or 100% ethanol will result in measurement failure or error. Be sure to use 95% ethanol where noted. It is common for palmitoyl carnitine to precipitate out of the 95% ethanol after thawing the frozen stock, thus causing large variability. Be sure to warm up the palmitoyl carnitine stock and vortex well prior to use. In addition, a highly variable pyruvate/malate assay result may be due to the pyruvate stock being &#62;2 weeks old. Be sure to remake frozen aliquots of pyruvate every 2 weeks. The assay run was modified to 2-min measurements to ensure mitochondrial state plateaus. If observation of exhaustion of ADP is desired, the researcher may extend the measurement time under the &#8220;Protocol&#8221; tab under the &#8220;Assay Wizard&#8221; forum. Finally, large variability occurs when the mitochondrial stock and mitochondria plus substrate solutions aren&#8217;t homogenized fully prior to loading. If variability between wells is high after the assay run, be sure to fully mix the substrate solution prior to the next experiment. Never vortex the mitochondria/substrate solutions, rather stir, mash, and gently triturate with a widened orifice pipette tip.
There are some limitations of this technique that are worth noting. First, the number of wells on the cell culture plate used for these assays is relatively low (i.e., 24 wells and at least 2 designated for blank wells). If it is desired to perform all 5 of these assays on one plate, the researcher is only able to examine the responses from one mouse at a time. However, it should be noted that 96 well instruments are available for higher throughput. Secondly, there are inherent strengths and weaknesses in assessing mitochondrial dysfunction in isolated mitochondria compared to in intact cells1. Some weaknesses include having less physiological relevance compared to intact cells and inducing artifacts from the isolation process. Finally, the success of this method is contingent on the quality of the mitochondrial isolation process.
Although some of these assays have been either developed in different systems or have been validated in other animal models, the methods presented herein are the first to synthesize all of the aforementioned assays for the optimal use in a multi-well oxygen consumption measurement machine using mouse skeletal muscle. Importantly, all 5 of these assays can be performed with the amount of mitochondria isolated from ~75 - 100 mg of mouse skeletal muscle, thus providing high throughput with minimal material. Of great significance, the ability of multi-well oxygen consumption technologies to perform assays with minimal quantities of mitochondria, combined with an optimized isolation method, allows the researcher to perform a multitude of other experiments with the remainder of skeletal muscle tissue (e.g., western blots, RT-PCR, enzymatic assays, etc.), which is often a struggle with this animal model.
In conclusion, the methods presented herein provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using minimal quantities of mouse skeletal muscle. Importantly, the presented methods require minimal quantities of tissue and mitochondria. Once mastered, the techniques described herein will allow researchers to determine a potential mechanism of a compound or gene product on mitochondrial oxygen consumption in a commonly used animal model.

The main physiological role of skeletal muscle mitochondria is to produce ATP from oxidative phosphorylation1. Importantly, skeletal muscle mitochondria play a specific role in exercise capacity2, aging3, degenerative disease4 and Type II diabetes5. As an aging society, and Type II Diabetes being the 7th leading cause of death in the United States6, the need for methods that assess mitochondrial function has become increasingly more prevalent in biomedical research7,8. Specifically, the measurement of oxygen consumption has exceptional utility in the assessment of mitochondrial function since it represents the coordinated function between mitochondrial and nuclear genomes to express functional components of oxidative phosphorylation9.
Several technologies exist that enable the measurement of oxygen consumption in intact cells and isolated mitochondria7-10. In addition, assays have been developed for multiple cell types and tissues, and in a variety of species that allow for the measurement of mitochondrial pathways and respiratory control9,11,12. However, there is currently a void in the literature in regards to the compilation of all of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based oxygen consumption technologies. Importantly, use of microplate based respirometric assays is growing among mitochondrial biologists and allows for high throughput measurements using minimal quantities of isolated mitochondria9. Therefore, a collection of microplate based respirometric assays were developed that allow pinpointing of where abnormalities and/or adaptations may be occurring in the electron transport chain (ETC). In addition, two additional microplate based respirometric assays were developed that enable the assessment of the coordination between the tricarboxylic acid (TCA) cycle and the ETC, and between &#223;-oxidation and the ETC. Importantly, the presented methods provide a clear and concise way to measure mechanistic changes in mitochondrial function in a commonly used animal model.

<table><tbody><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td>Store at RT</td></tr><tr><td>D-Mannitol</td><td>Sigma Aldrich</td><td>63559</td><td>Store at RT</td></tr><tr><td>Potassium phosphate monobasic, minimum 99.0%</td><td>Sigma Aldrich</td><td>P5379</td><td>Store at RT</td></tr><tr><td>Magnesium chloride hexahydrate, ACS reagent, 99.0-102.0%</td><td>Sigma Aldrich</td><td>M9272</td><td>Store at RT</td></tr><tr><td>HEPES minimum 99.5% titration</td><td>Sigma Aldrich</td><td>H3375</td><td>Store at RT</td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td>Store at RT</td></tr><tr><td>Essentially Fatty&amp;nbsp;</td><td>Sigma Aldrich</td><td>A6003</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Acid Free- BSA</td><td></td><td></td><td></td></tr><tr><td>Pyruvic Acid, 98%</td><td>Sigma Aldrich</td><td>107360</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Succinic Acid</td><td>Sigma Aldrich</td><td>S9512</td><td>Store at RT</td></tr><tr><td>L(-) Malic Acid, BioXtra, &amp;ge;95%</td><td>Sigma Aldrich</td><td>M6413</td><td>Store at RT</td></tr><tr><td>L-Glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td>Store at RT</td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethyl-p-phenylenediamine</td><td>Sigma Aldrich</td><td>T7394</td><td>Store at RT</td></tr><tr><td>99%, powder [TMPD]</td><td></td><td></td><td></td></tr><tr><td>Palmitoyl L-carnitine chloride</td><td>Sigma Aldrich</td><td>P1645</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Oligomycin A, &amp;ge; 95% (HPLC)</td><td>Sigma Aldrich</td><td>75351</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Carbonyl cyanide 4-(trifluoromethoxy)</td><td>Sigma Aldrich</td><td>C2920</td><td>Store at 2 - 8 &amp;deg;C</td></tr><tr><td>phenylhydrazone</td><td></td><td></td><td></td></tr><tr><td>&amp;ge;98% (TLC), powder [FCCP]</td><td></td><td></td><td></td></tr><tr><td>Antimycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Adenosine 5&amp;prime;-diphosphate monopotassium salt dehydrate [ADP]</td><td>Sigma Aldrich</td><td>A5285</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td>Store at RT</td></tr><tr><td>Pierce&amp;trade; BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>23225</td><td>N/A</td></tr></tbody></table>

using isolated mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

Figure 1 represents oxygen consumption rates (OCR) for the pyruvate/malate, succinate/rotenone, palmitoyl carnitine/malate, and glutamate/malate assays (Coupling Assays). These assay tracings are displayed as Oxygen Consumption Rate, or OCR, vs. time, are background correceted, and are displayed as point-to-point rates. Each panel represents oxygen consumption in different mitochondrial states as described by Chance and Williams14. The first panel represents basal oxygen consumption, or State 2. The second panel, after injection of ADP, represents maximal coupled respiration, or State 3. The third panel, after injection of oligomycin A (an inhibitor of Complex V), represents respiration due to proton leak, or State 4o. The fourth panel, after injection of FCCP, represents maximal uncoupled respiration, or State 3u. Finally, the fifth panel, after injection of Antimycin A, represents the inhibition of oxidative respiration. Notably, all mitochondrial states have minimal standard deviation. This is due to thorough mixing of the mitochondrial stock and the mitochondria/substrate mixes, and the attainment of a single monolayer of mitochondria after the adherence spin (Step 2.6). On the other hand, loading unequal mitochondria in each well and not attaining a single monolayer of mitochondria in the well of the microplate leads to increased standard deviation in each state as displayed in Figure 2.
The tracings in Figure 1 display plateaus for each mitochondrial state and for each substrate. The plateau attained after two measurement cycles suggests good mitochondrial quality and that the mitochondria remain adhered to the well throughout the duration of the assay. In addition, the attainment of plateaued maximal rates may be more desirable since this allows the researcher to take the average OCR at these mitochondrial states, thus reducing bias that may occur by arbitrarily selecting a point.
The amount of mitochondria per well was determined by optimization trials. The optimal amount of mitochondria per well should result in State 2 rates between 100-200 pmol/min/well and state 3 rates &#60; 1,500 pmol/min/well, since these values are within the dynamic oxygen sensing range of the multi-well oxygen consumption measurement machine. Loading too much mitochondrial protein per well may result in OCRs beyond the dynamic range of the instrument (Figure 3A). Figure 3 depicts loading 3.5 &#181;g of mitochondrial protein per well (blue tracing) compared to loading 2.5 &#181;g of mitochondrial protein per well (red tracing) for the succinate/rotenone assay. Loading too much mitochondrial protein per well can also lead to the exhaustion of oxygen within the microchamber of the well, thus preventing accurate measurement of OCR for each successive measurement9 (Figure 3B). Point-to-point OCR is the&#160;instantaneous rate of change of the OCR. If flat, the OCR is steady/stable, but if decreasing, then there may be a biologic or technical issue. The steep decline in OCR in State 3 and State 3u respiration (Figure 3A) is caused by the mitochondria exhausting the oxygen supply before the end of the measurement (Figure 3B).
Figure 4 represents OCR vs. time for the electron flow assay. The tracing is corrected and displayed as described for Figure 1. The first panel in this assay represents State 3u respiration on pyruvate/malate via Complex I. The second panel, after injection of rotenone, represents inhibition of Complex I mediated respiration. The third panel, after injection of succinate, represents substrate stimulated State 3u with electrons entering the ETC at Complex II (Complex II mediated respiration). The fourth panel, after injection of Antimycin A, represents inhibition of Complex III and thus total respiration. Finally, the fifth panel, after injection of ascorbate/TMPD, represents Complex IV mediated respiration. Similar to the tracings in Figure 1, all mitochondrial states have minimal standard deviation, and each rate has or nearly has attained a plateau.
<img alt="Figure 1" src="/files/ftp_upload/53216/53216fig1.jpg" /> 
Figure 1.&#160;Coupling Assays. (A) 10 mM Pyruvate/ 5 mM malate, (B) 10 mM succinate/ 2 &#181;M rotenone, (C) 40 &#181;M palmitoyl carnitine/ 1 mM malate, and (D) 10 mM glutamate/ 10 mM malate coupled mitochondrial respiration assay tracings as determined by multi-well measurement of oxygen consumption. Values are expressed as mean &#177; SD. Mitochondrial protein loaded per well was 3.5 &#181;g for all assays except succinate/rotenone, which utilizes 2.5 &#181;g of mitochondrial protein per well. Data represents n=3 paired biological replicates. OCR = Oxygen Consumption Rate; ADP = Adenosine diphosphate; Oligo = Oligomycin A; FCCP = Carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone; Anti-A = Antimycin A; PYR = Pyruvate; SUCC = Succinate; ROT = Rotenone; PAL-C = palmitoyl carnitine; GLUT = glutamate. &#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53216/53216fig1large.png" style="font-size: 14px; line-height: 28px;" target="_blank">Please click here to view a larger version of this figure.</a>&#160;
<img alt="Figure 2" src="/files/ftp_upload/53216/53216fig2.jpg" /> 
Figure 2.&#160;Highly Variable Pyruvate/Malate Assay. Highly variable 10 mM pyruvate/ 5 mM malate assay caused by incompletely mixing mitochondria from the mitochondrial stock in the substrate/MAS mix, thus leading to variable mitochondrial protein loading in each well. Mitochondrial protein loaded per well was 3.5 &#181;g. Data represents n=3 paired biological replicates. OCR=Oxygen Consumption Rate; ADP= Adenosine diphosphate; Oligo= Oligomycin A; FCCP= Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; Anti-A= Antimycin A; PYR=Pyruvate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53216/53216fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53216/53216fig3.jpg" /> 
Figure 3.&#160;Overloading Mitochondrial Protein for the Succinate/Rotenone Assay. (A) Oxygen consumption rates outside the dynamic range of the multi-well oxygen consumption measurement machine caused by loading 3.5 &#181;g of mitochondrial protein per well (blue tracing) compared to loading 2.5 &#181;g of mitochondrial protein per well (red tracing). (B) Oxygen tension approaching zero following ADP and FCCP injections caused by loading excessive mitochondrial protein (3.5 &#181;g) per well (blue tracing) compared to loading an optimal amount (2.5 &#181;g) of mitochondrial protein per well (red tracing). Data represents n = 3 paired biological replicates per mitochondrial protein amount. OCR = Oxygen Consumption Rate; ADP = Adenosine diphosphate; Oligo = Oligomycin A; FCCP = Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; Anti-A = Antimycin A; SUCC = Succinate; ROT = Rotenone; O2 = Oxygen; mm Hg = millimeters of mercury. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53216/53216fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53216/53216fig4.jpg" /> 
Figure 4.&#160;Electron Flow Assay. 5 mM pyruvate/ 1 mM malate+ 4 &#181;M FCCP, electron flow mitochondrial respiration assay tracing as determined by multi-well measurement of oxygen consumption. Values are expressed as mean &#177; SD. Mitochondrial protein loaded per well was 3.5 &#181;g. Data represents n=3 paired biological replicates. OCR = Oxygen Consumption Rate; Anti-A = Antimycin A; Asc = Ascorbate; TMPD = N,N,N&#8242;,N&#8242;-tetramethyl-p-phenylenediamine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53216/53216fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock Concentration&#160;</td>
			<td>MW</td>
			<td>Final Volume</td>
			<td>Mass Added</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td></td>
			<td>(M)</td>
			<td>(g/mol)</td>
			<td>(ml)</td>
			<td>(g or ml)</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA, pH 7.2</td>
			<td>0.1</td>
			<td>380.35</td>
			<td>100 ml of 1M Tris Base</td>
			<td>3.801 g</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1</td>
			<td>238.3</td>
			<td>250 ml&#160;&#160;of DiH2O</td>
			<td>59. 57 g</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>MgCl2, hexahydrate</td>
			<td>1</td>
			<td>203.31</td>
			<td>250 ml&#160;&#160;of DiH2O</td>
			<td>50.82 g</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Pyruvate pH 7.4</td>
			<td>0.1</td>
			<td>88.06 (Comes as 14.11 M solution)</td>
			<td>40 ml&#160;&#160;of DiH2O</td>
			<td>0.283 ml of pyruvic acid</td>
			<td>Make 1 ml aliquots and store at -20 &#176;C, make fresh every two weeks</td>
		</tr>
		<tr>
			<td>Succinate pH 7.4</td>
			<td>0.5</td>
			<td>118.09</td>
			<td>100 ml&#160;&#160;of DiH2O</td>
			<td>9.4 g of succinic acid</td>
			<td>Make 1 ml aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Malate, pH 7.4</td>
			<td>0.5</td>
			<td>134.09</td>
			<td>100 ml&#160;&#160;of 95% Ethanol</td>
			<td>6.7 g of malic acid</td>
			<td>Make 200 &#956;l aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>TMPD</td>
			<td>0.01</td>
			<td>164.25</td>
			<td>10&#160;ml&#160;</td>
			<td>0.0164 g</td>
			<td>Make 300 &#956;l&#160;&#160;aliquots and store at -20 &#176;C; Mix with an equimolar amount of ascorbate to keep TMPD reduced</td>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>0.01</td>
			<td>436.07</td>
			<td>1.14 ml&#160;&#160;of 95% Ethanol</td>
			<td>0.005 g</td>
			<td>Make 40 &#956;l&#160;&#160;aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A</td>
			<td>0.006</td>
			<td>791.06</td>
			<td>0.987 ml&#160;&#160;of 95% Ethanol</td>
			<td>0.005 g</td>
			<td>Make 20 &#956;l&#160;&#160;aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>0.01</td>
			<td>254.17</td>
			<td>3.9 ml&#160;&#160;of 95% Ethanol</td>
			<td>0.01 g</td>
			<td>Make 40 &#956;l&#160;&#160;aliquots and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>0.001</td>
			<td>394.4</td>
			<td>10 ml&#160;&#160;of 95% Ethanol</td>
			<td>0.0039&#160; g</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>0.005</td>
			<td>548.63</td>
			<td>9.12 ml&#160;&#160;of 95% Ethanol</td>
			<td>0.025 g</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>K+ ADP</td>
			<td>0.5</td>
			<td>501.32</td>
			<td>3.9 ml&#160;&#160;of DiH2O</td>
			<td>1.0 g&#160;</td>
			<td>Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Malic Acid, pH 7.4</td>
			<td>0.5</td>
			<td>134.09</td>
			<td>40&#160;ml&#160;</td>
			<td>2.68 g</td>
			<td>Make 200 &#956;l&#160;&#160;aliquots and store at -20 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;Stock Solutions
<table border="0">
	<tbody>
		<tr>
			<td rowspan="2">Reagent</td>
			<td>Stock Concentration&#160;</td>
			<td>Mass Added</td>
			<td rowspan="2">Final Molarity/Percent</td>
		</tr>
		<tr>
			<td>(M)</td>
			<td>(g or ml)</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>--</td>
			<td>11.98 g</td>
			<td>70 mM</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>--</td>
			<td>20.04 g</td>
			<td>220 mM</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>--</td>
			<td>0.34 g</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>MgCl2, hexahydrate</td>
			<td>1</td>
			<td>2.5&#160;ml&#160;</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>1</td>
			<td>1.0&#160;ml&#160;</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.1</td>
			<td>5.0&#160;ml&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Essentially Fatty Acid Free- BSA</td>
			<td>--</td>
			<td>&#160;1.0 g</td>
			<td>0.20%</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;MAS Mix. pH 7.4, 500 ml: Aliquot 25 ml and store at -20 &#186;C *Note: Exclude BSA for MAS mix used for assay injections.
<table border="0">
	<tbody>
		<tr>
			<td>Substrate Medium&#160;</td>
			<td>Final Concentration</td>
			<td>Amount of stock (&#181;l)</td>
			<td>Amount of MAS* (ml)</td>
		</tr>
		<tr>
			<td rowspan="2">Pyruvate/Malate</td>
			<td rowspan="2">10 mM/5 mM</td>
			<td>Pyruvate: 1,000</td>
			<td rowspan="2">9</td>
		</tr>
		<tr>
			<td>Malate: 100</td>
		</tr>
		<tr>
			<td rowspan="2">Succicinate/Rotenone</td>
			<td rowspan="2">10 mM/2 &#181;M</td>
			<td>Succinate: 200</td>
			<td rowspan="2">10</td>
		</tr>
		<tr>
			<td>Rotenone 20</td>
		</tr>
		<tr>
			<td rowspan="3">*Pyruvate/Malate + FCCP&#160;</td>
			<td rowspan="3">5 mM/1 mM/4 &#181;M</td>
			<td>Pyruvate: 500</td>
			<td rowspan="3">10</td>
		</tr>
		<tr>
			<td>FCCP: 4</td>
		</tr>
		<tr>
			<td>Malate: 20</td>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine/Malate</td>
			<td>40 &#181;M/1 mM</td>
			<td>Palmitoylcarnitine: 40 Malate: 20</td>
			<td>10</td>
		</tr>
		<tr>
			<td rowspan="2">Glutamate/Malate</td>
			<td rowspan="2">10 mM/10 mM</td>
			<td>Glutamate: 400</td>
			<td rowspan="2">10</td>
		</tr>
		<tr>
			<td>Malate: 200</td>
		</tr>
	</tbody>
</table>
Table 3.&#160;Substrate Solutions pH 7.4: Make fresh the day of the experiment. *Electron flow assay solution.
<table border="0">
	<tbody>
		<tr>
			<td>Injection Medium&#160;</td>
			<td>Concentration</td>
			<td>Amount of stock&#160; (&#181;l)</td>
			<td>Amount of MAS (ml)</td>
			<td>Amount injected into Cartridge&#160;</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>(After injected in plate)</td>
		</tr>
		<tr>
			<td>ADP&#160;</td>
			<td>50 mM</td>
			<td>300 &#181;l</td>
			<td>3</td>
			<td>50&#160;&#181;l</td>
			<td>5.0 mM</td>
		</tr>
		<tr>
			<td>Oligomycin A&#160;</td>
			<td>20 &#181;M</td>
			<td>10&#160;&#181;l</td>
			<td>3</td>
			<td>55&#160;&#181;l</td>
			<td>2.0 &#181;M</td>
		</tr>
		<tr>
			<td>FCCP&#160;</td>
			<td>40 &#181;M</td>
			<td>12&#160;&#181;l</td>
			<td>3</td>
			<td>60&#160;&#181;l</td>
			<td>4.0 &#181;M</td>
		</tr>
		<tr>
			<td>Antimycin A&#160;</td>
			<td>40 &#181;M</td>
			<td>24&#160;&#181;l</td>
			<td>3</td>
			<td>65&#160;&#181;l</td>
			<td>4.0 &#181;M</td>
		</tr>
	</tbody>
</table>
Table 4.&#160;Injections for Coupled Assays. pH 7.4: Make fresh the day of the experiment. *Coupling assays include (but are not limited to) pyruvate/malate, succinate/rotenone, palmitoyl carnitine/malate, and glutamate/malate.
<table border="0">
	<tbody>
		<tr>
			<td>Injection Medium&#160;</td>
			<td>Concentration</td>
			<td>Amount of stock (g or ul)</td>
			<td>Amount of MAS (ml)</td>
			<td>Amount injected into Cartridge&#160;</td>
			<td>Final Concentration (After injected in plate)</td>
		</tr>
		<tr>
			<td>Rotenone&#160;</td>
			<td>20 &#181;M</td>
			<td>60&#160;&#181;l</td>
			<td>3</td>
			<td>50&#160;&#181;l</td>
			<td>2.0 &#181;M</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>50 mM</td>
			<td>300&#160;&#181;l</td>
			<td>3</td>
			<td>55&#160;&#181;l</td>
			<td>5.0 mM</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>40 &#181;M</td>
			<td>24&#160;&#181;l</td>
			<td>3</td>
			<td>60&#160;&#181;l</td>
			<td>4.0 &#181;M</td>
		</tr>
		<tr>
			<td>TMPD/Ascorabte</td>
			<td>1 mM, 100 mM</td>
			<td>TMPD : 300&#160;&#181;l</td>
			<td>3</td>
			<td>65&#160;&#181;l</td>
			<td>100 &#181;M, 10mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>Ascorbate: 0.059 g</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 5.&#160;Injections for Electron Flow Assay. pH 7.4: Make fresh the day of the experiment
<table border="0">
	<tbody>
		<tr>
			<td>Command</td>
			<td>Time (min)</td>
			<td># of cycles</td>
		</tr>
		<tr>
			<td>Calibrate</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wait</td>
			<td>10 min (to allow plate to warm from&#160; adherence step)</td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1 min</td>
			<td rowspan="2">2</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Inject A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1 min</td>
			<td rowspan="2">2</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Inject B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1 min</td>
			<td rowspan="2">2</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Inject C</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1 min</td>
			<td rowspan="2">2</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>2 min</td>
		</tr>
		<tr>
			<td>Inject D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mix</td>
			<td>1 min</td>
			<td rowspan="2">2</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>2 min</td>
		</tr>
	</tbody>
</table>
Table 6.&#160;Instrument Run Protocol.

Watch this Scientific Journal Video about Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements at JoVE.com

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

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.6K Views.  Virginia Tech. 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.

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

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

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

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Medicine

Mitochondrial Isolation from Skeletal Muscle

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

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

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

Biology

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

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

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

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

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

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

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

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

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

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

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

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

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