We wish to thank Juan J. Tena for the use of the homogenizer and the CABD Proteomics and Animal Husbandry facilities for technical support. This work was supported by the Spanish Ministry of Education, Culture and Sports through fellowship FPU16/03264 to J.D.H.C., the Association Fran&#231;aise contre les Myopathies (AFM) through fellowship grant #22450 to C.V.-G., an Institutional Grant MDM-2016-0687 (Maria de Maeztu Excellence Unit, Department of Gene Regulation and Morphogenesis at CABD) and BFU2017-83150-P to J.J.C. The Junta de Andaluc&#237;a grant P18-RT-4572, the FEDER Funding Program from the European Union, and Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T to P.N.

Mouse housing and tissue collection were performed using protocols approved by the Universidad Pablo de Olavide Ethics Committee (Sevilla, Spain; protocols 24/04/2018/056 and 12/03/2021/033) in accordance with Spanish Royal Decree 53/2013, European Directive 2010/63/EU, and other relevant guidelines.
1. Preparation of stocks, buffers, and reagents for the respiration assays
<ol>
	<li>Prepare the following stock solutions, which can be stored at the indicated temperature for ...

<ol>
	<li>Spinelli, J. B., Haigis, 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+multifaceted+contributions+of+mitochondria+to+cellular+metabolism.">The multifaceted contributions of mitochondria to cellular metabolism.</a> Nature Cell Biology. 20 (7), 745-754 (2018).</li><li>Alberts, B., 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=The+mitochondrion.">The mitochondrion.</a> Molecular Biology of the Cell, 4th edition. , (2002).</li><li>Turunen, M., Olsson, J., Dallner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+function+of+coenzyme+Q.">Metabolism and function of coenzyme Q.</a> Biochimica et Biophysica Acta. 1660 (1-2), 171-199 (2004).</li><li>Alc&#225;zar-Fabra, M., Trevisson, E., Brea-Calvo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+syndromes+associated+with+coenzyme+Q10+deficiency.">Clinical syndromes associated with coenzyme Q10 deficiency.</a> Essays in Biochemistry. 62 (3), 377-398 (2018).</li><li>Banerjee, R., Purhonen, J., Kallij&#228;rvi, J. <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+coenzyme+Q+junction+and+complex+III:+biochemistry+and+pathophysiology.">The mitochondrial coenzyme Q junction and complex III: biochemistry and pathophysiology.</a> The FEBS Journal. , (2021).</li><li>Gorman, G. 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=Mitochondrial+diseases.">Mitochondrial diseases.</a> Nature Reviews. Disease Primers. 2, 16080 (2016).</li><li>Villalba, J. M., Navas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+coenzyme+Q+biosynthesis+pathway+in+eukaryotes.">Regulation of coenzyme Q biosynthesis pathway in eukaryotes.</a> Free Radical Biology &amp; Medicine. 165, 312-323 (2021).</li><li>Li, Z., Graham, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+oxygen+consumption+using+a+Clark+electrode.">Measurement of mitochondrial oxygen consumption using a Clark electrode.</a> Methods in Molecular Biology. 837, 63-72 (2012).</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 (7), 21746 (2011).</li><li>Pala, F., 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=Distinct+metabolic+states+govern+skeletal+muscle+stem+cell+fates+during+prenatal+and+postnatal+myogenesis.">Distinct metabolic states govern skeletal muscle stem cell fates during prenatal and postnatal myogenesis.</a> Journal of Cell Science. 131 (14), 212977 (2018).</li><li>Shintaku, 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=MyoD+regulates+skeletal+muscle+oxidative+metabolism+cooperatively+with+alternative+NF-&#312;B.">MyoD regulates skeletal muscle oxidative metabolism cooperatively with alternative NF-&#312;B.</a> Cell Reports. 17 (2), 514-526 (2016).</li><li>Li, R., 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=Development+of+a+high-throughput+method+for+real-time+assessment+of+cellular+metabolism+in+intact+long+skeletal+muscle+fibre+bundles.">Development of a high-throughput method for real-time assessment of cellular metabolism in intact long skeletal muscle fibre bundles.</a> The Journal of Physiology. 594 (24), 7197-7213 (2016).</li><li>Schuh, R. A., Jackson, K. C., Khairallah, R. J., Ward, C. W., Spangenburg, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+respiration+in+intact+single+muscle+fibers.">Measuring mitochondrial respiration in intact single muscle fibers.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 302 (6), 712-719 (2012).</li><li>Rosenblatt, J. D., Lunt, A. I., Parry, D. J., Partridge, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+satellite+cells+from+living+single+muscle+fiber+explants.">Culturing satellite cells from living single muscle fiber explants.</a> In Vitro Cellular &amp; Developmental Biology. Animal. 31 (10), 773-779 (1995).</li><li>Keire, P., Shearer, A., Shefer, G., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+skeletal+muscle+myofibers+as+a+means+to+analyze+satellite+cells.">Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells.</a> Methods in Molecular Biology. 946, 431-468 (2013).</li><li>Shintaku, J., Guttridge, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+aerobic+respiration+in+intact+skeletal+muscle+tissue+by+microplate-based+respirometry.">Analysis of aerobic respiration in intact skeletal muscle tissue by microplate-based respirometry.</a> Methods in Molecular Biology. 1460, 337-343 (2016).</li><li>Bharadwaj, M. 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=Preparation+and+respirometric+assessment+of+mitochondria+isolated+from+skeletal+muscle+tissue+obtained+by+percutaneous+needle+biopsy.">Preparation and respirometric assessment of mitochondria isolated from skeletal muscle tissue obtained by percutaneous needle biopsy.</a> Journal of Visualized Experiments: JoVE. (96), e52350 (2015).</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. (49), e2452 (2011).</li><li>Iuso, A., Repp, B., Biagosch, C., Terrile, C., Prokisch, H. <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+bioenergetics+in+isolated+mitochondria+from+various+mouse+tissues+using+Seahorse+XF96+analyzer.">Assessing mitochondrial bioenergetics in isolated mitochondria from various mouse tissues using Seahorse XF96 analyzer.</a> Methods in Molecular Biology. 1567, 217-230 (2017).</li><li>Divakaruni, A. S., Rogers, G. W., Murphy, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mitochondrial+function+in+permeabilized+cells+using+the+Seahorse+XF+analyzer+or+a+Clark-type+oxygen+electrode.">Measuring mitochondrial function in permeabilized cells using the Seahorse XF analyzer or a Clark-type oxygen electrode.</a> Current Protocols in Toxicology. 60, 1-16 (2014).</li><li>Das, K. C., Muniyappa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+mitochondrial+energy+dynamics+in+the+mice+heart:+role+of+superoxide+dismutase-2.">Age-dependent mitochondrial energy dynamics in the mice heart: role of superoxide dismutase-2.</a> Experimental Gerontology. 48 (9), 947-959 (2013).</li><li>Aw, W. C., Bajracharya, R., Towarnicki, S. G., Ballard, J. W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+bioenergetic+functions+from+isolated+mitochondria+in+Drosophila+melanogaster.">Assessing bioenergetic functions from isolated mitochondria in Drosophila melanogaster.</a> Journal of Biological Methods. 3 (2), 42 (2016).</li><li>Sakamuri, S. S. V. P., 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=Measurement+of+respiratory+function+in+isolated+cardiac+mitochondria+using+Seahorse+XFe24+analyzer:+applications+for+aging+research.">Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 analyzer: applications for aging research.</a> Gerontology. 40 (3), 347-356 (2018).</li><li>Boutagy, N. E., Pyne, E., Rogers, G. W., Ali, M., Hulver, M. W., Frisard, M. I. <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+from+minimal+quantities+of+mouse+skeletal+muscle+for+high+throughput+microplate+respiratory+measurements.">Isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements.</a> Journal of Visualized Experiments: JoVE. (105), e53217 (2015).</li><li>Sperling, J. 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=Measuring+respiration+in+isolated+murine+brain+mitochondria:+implications+for+mechanistic+stroke+studies.">Measuring respiration in isolated murine brain mitochondria: implications for mechanistic stroke studies.</a> Neuromolecular Medicine. 21 (4), 493-504 (2019).</li><li>Boutagy, N. E., Rogers, G. W., Pyne, E. S., Ali, M. M., Hulver, M. W., Frisard, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+isolated+mitochondria+from+minimal+quantities+of+mouse+skeletal+muscle+for+high+throughput+microplate+respiratory+measurements.">Using isolated mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements.</a> Journal of Visualized Experiments: JoVE. (105), e53216 (2015).</li></ol>

The authors declare that they have no conflicts of interest to disclose.

All methods used to study mitochondrial respiration have their limitations; hence, it is crucial to select the method that best suits a specific experimental question. This work provides an updated and detailed protocol to isolate mitochondria from mouse skeletal muscle to perform different respiratory assays to investigate mitochondrial function. Indeed, the study of mitochondrial bioenergetics in isolated mitochondria using microplate-based technologies is valuable to study tissue-specific respiration in terms of repro...

Mitochondria are the primary metabolic organelles in the cell1. These specialized membrane-enclosed organelles use nutrient molecules to produce energy in the form of adenosine triphosphate (ATP) by OXPHOS. This process relies on the transfer of electrons from donor molecules in a series of redox reactions in the ETC2. CoQ is the only redox-active lipid that is endogenously produced in all cellular membranes and circulating lipoproteins that shows antioxidant function3. It is an essential component of the ETC, transferring electrons from NADH-dependent complex I and FADH2-dependent complex II to complex III, although many other reductases can drive the reduction of mitochondrial CoQ to ubiquinol as a mandatory step in multiple cellular metabolic pathways4,5.
Throughout the process, an electrochemical proton gradient is created across the mitochondrial inner membrane, which is transformed into biologically active energy by the ATP synthase complex V2. Consequently, mitochondrial dysfunction leads to a myriad of pathological conditions mainly affecting tissues with high-energy requirements-the brain, heart, and skeletal muscle6,7. Therefore, it is fundamental to develop methods to accurately analyze mitochondrial bioenergetics to investigate its role in health and disease, particularly in highly energetic tissues such as skeletal muscles.
The Clark-type oxygen electrode has been classically used in the study of mitochondrial respiration8. However, this system has been progressively displaced by higher-resolution technologies, with microplate-based oxygen consumption technologies such as Agilent Seahorse XF analyzers being especially popular9. In the skeletal muscle field, these studies are typically conducted in cultured cells, mainly in the C2C12 immortalized mouse myoblast cell line or primary cultures derived from satellite cells10,11. However, these studies do not fully recapitulate the situation in vivo, especially when investigating mitochondrial biology and function at the tissue level upon specific insults, nongenetic interventions, or genetic manipulations.
Furthermore, respiration assays in cells are more complex due to additional factors, including extra-mitochondrial demand of ATP and assay substrates or signaling events, which could mislead the interpretation of the results. Alternatively, it is also possible to use single or bundles of freshly isolated myofibers from muscles. However, the isolation method is technically challenging and only feasible for a few muscle types. In this case, flexor digitorum brevis (FDB) and extensor digitorum longus (EDL) muscles are mainly used10,12,13, although a few reports describe the use of other muscle types as well14,15.
Bioenergetic profiling of skeletal muscle sections has also been reported16. The major advantage of this method is that intact muscles can be studied (the authors show that slicing through fibers does not disturb results when compared with isolated myofibers). However, mitochondrial access to substrates and assay inhibitors is limited, and thus, only a few parameters can be measured16. Finally, isolated mitochondria can be likewise employed9,17,18,19. In this case, mitochondria lose their cytosolic environment, which could affect their function. In contrast, this method guarantees access to substrates and inhibitors, enables the analysis of a plethora of sample types, and typically requires less material.
This paper describes a method to perform the bioenergetic profiling of isolated mitochondria from mouse skeletal muscle using microplate-based respirometric assays (Figure 1). In particular, three protocols are detailed: the Coupling Assay, CA to assess the degree of coupling between the ETC and the OXPHOS machinery; the Electron Flow Assay, EFA to measure the activity of the individual ETC complexes; and the BOX assay to determine mitochondrial &#946;-oxidation capacity. Notably, only small amounts of samples are required compared with conventional respirometry methods. The isolation protocol used here has been modified from the method published elsewhere18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ADP</td>
			<td>Sigma</td>
			<td>A5285</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>AKT antibody</td>
			<td>Cell Signaling Technology</td>
			<td>C67E7</td>
			<td>Rabbit (Host species)</td>
		</tr>
		<tr>
			<td>anti-Goat HRP</td>
			<td>Sigma</td>
			<td>401504</td>
			<td>Rabbit (Host species)</td>
		</tr>
		<tr>
			<td>anti-Mouse HRP</td>
			<td>Cell Signaling</td>
			<td>#7076</td>
			<td>Horse (Host species)</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma</td>
			<td>A8674</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>anti-Rabbit HRP</td>
			<td>Cell Signaling</td>
			<td>#7074</td>
			<td>Goat (Host species)</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A5960</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Bactin antibody</td>
			<td>Sigma</td>
			<td>MBS4-48085</td>
			<td>Goat (Host species)</td>
		</tr>
		<tr>
			<td>Bio-Rad Protein Assay Kit II</td>
			<td>Bio-Rad</td>
			<td>5000002</td>
			<td>It includes 5x Bradford reagent and BSA of known concentration for the standard curve</td>
		</tr>
		<tr>
			<td>BSA, fraction V, Fatty Acid-Free</td>
			<td>Calbiochem</td>
			<td>126575</td>
			<td>Stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>C tube</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>Purple lid</td>
		</tr>
		<tr>
			<td>Calnexin antibody</td>
			<td>ThermoFisher</td>
			<td>MA3-027</td>
			<td>Mouse (Host species)</td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>BDH</td>
			<td>280254D</td>
			<td>Stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E-4378</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma</td>
			<td>C2920</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Homogenizer</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>HSP70 antibody</td>
			<td>Proteintech</td>
			<td>10995-1-AP</td>
			<td>Rabbit (Host species)</td>
		</tr>
		<tr>
			<td>LDH-A antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC27230</td>
			<td>Goat (Host species)</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>ChemCruz</td>
			<td>sc-255260A</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Malic acid</td>
			<td>Sigma</td>
			<td>P1645</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>BMG LABTECH GmbH</td>
			<td>POLARstar OMEGA S/N 415-0292</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Milli-Q water</td>
			<td>Millipore system</td>
			<td>F7HA17757A</td>
			<td>Ultrapure water</td>
		</tr>
		<tr>
			<td>mtTFA antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC23588</td>
			<td>Goat (Host species)</td>
		</tr>
		<tr>
			<td>Na+/K+-ATPase &#945;1 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB300-14755</td>
			<td>Mouse (Host species)</td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma</td>
			<td>O4876</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Palmitoyl-L-carnitine</td>
			<td>Sigma</td>
			<td>P1645</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Sigma</td>
			<td>P4417-100TAB</td>
			<td>1x stock at RT</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>ChemCruz</td>
			<td>sc-203211</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma</td>
			<td>60377</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Pyruvic acid</td>
			<td>Sigma</td>
			<td>107360</td>
			<td>Stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma</td>
			<td>R8875</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Seahorse XF24 mitochondrial flux analyzer</td>
			<td>Agilent Technologies</td>
			<td>420179</td>
			<td>XFe24 model</td>
		</tr>
		<tr>
			<td>Seahorse XFe24 FluxPak mini</td>
			<td>Agilent Technologies</td>
			<td>102342-100</td>
			<td>The kit includes cartridges, microplates, and calibrant solution</td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>Sigma</td>
			<td>S7626</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S9378</td>
			<td>Stock at RT</td>
		</tr>
		<tr>
			<td>TIMM23 antibody</td>
			<td>Abcam</td>
			<td>ab230253</td>
			<td>Rabbit (Host species)</td>
		</tr>
		<tr>
			<td>TMPD</td>
			<td>Sigma</td>
			<td>T7394</td>
			<td>Stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>TOMM20 antibody</td>
			<td>Abcam</td>
			<td>ab56783</td>
			<td>Mouse (Host species)</td>
		</tr>
		<tr>
			<td>VDAC antibody</td>
			<td>Abcam</td>
			<td>ab15895</td>
			<td>Rabbit (Host species)</td>
		</tr>
	</tbody>
</table>

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.

The protocol presented here allows the in vivo analysis of mitochondrial respiration through the isolation of mitochondria from mouse skeletal muscle. An outline of the method is shown in Figure 1. After dissecting skeletal muscles from the hindlimbs (Figure 2), tissues are homogenized and mitochondria purified, under isotonic conditions, through serial centrifugations. The purity of the different fractions obtained during the isolation process can be a...

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Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

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 ...

This word describes respiration analysis in processing mitochondria from mouse skeletal muscle. This protocol considerably reduce purification time and allows multiple samples to be processed simultaneously. Mitochondria from animals at any eight gender of United background can be mutilated using this protocol enabling the study of respiration in a wide variety of experimental conditions.This method is suitable for all research areas where mitochondria function is relevant including aging, drug testing, cancer, or development. It can even be adapted to other tissues and organisms as well. Ensure all materials are cold by placing everything on the ice during the procedure to protect mitochondria from damage.Place three 50 milliliter beakers per sample on ice and add 10 milliliters of PBS in beaker one, 10 milliliters of 10 millimolar EDTA PBS in beaker two, and four milliliters IB1 in beaker three. Spray the right hind limb of a euthanized mouse with 70%ethanol to prevent fur from shedding and tape the limb to the cork dissection board then tape the left fore limb. Make an incision with a sterile disposable scalpel through the skin from knee to toe.Grip the skin with toothed forceps at ankle level and cut it with fine scissors around the ankle. Ease the skin away from the underlying musculature with fine tip tweezers in one hand while pulling it up with toothed forceps in the other hand. Dissect all skeletal muscles from ankle to knee and remove all connective tissue over the tibialis anterior muscle using fine tweezers and scissors.Find the four distal tendons of the extensor digitorum longus muscle and section them close to their insertions in the toes. Locate the tibialis anterior distal tendon and cut it close to its insertion below the ankle. Pull the tibialis anterior and extensor digitorum longus tendons above the ankle to liberate the loose ends.Grip the loose ends of the tendons and ease the muscles away from the rest of the musculature in bones by pulling them up. Cut the proximal tendons of these muscles as close as possible to the kneecap. Turn the mouse upside down to proceed with gastrocnemius and soleus muscle extraction.Grip the pocket formed between the biceps, femoris, and the gastrocnemius and separate these muscles using fine scissors to visualize the proximal gastrocnemius tendon. Grip and carefully cut the distal Achilles tendon with fine scissors. Cut the proximal gastrocnemius tendon liberating it with the underlying soleus.Carefully dissect the remaining muscles and re-pin the mouse in the initial position to dissect the quadriceps muscle. Discard the adipose tissue then insert fine tweezers between the quadriceps and the femur to separate the muscle from the bone. Grip the distal quadriceps muscle tendons and cut the tendon as close as possible to the kneecap.Pull the quadriceps up and liberate them with fine scissors at the proximal insertion. Repeat the same for the left hind limb. Rinse all the muscles in beaker one, then two, followed by beaker three.Finally, mince all the muscles in beaker three with sharp scissors on ice. Transfer the suspension to a C tube, tightly close it, and attach it upside down onto the sleeve of the homogenizer. Divide the homogenate into two 2 milliliter pre-chilled micro centrifuge tubes and centrifuge for 10 minutes.Then transfer the supernatant to new pre-chilled tubes and centrifuge for another 10 minutes. Combine the pellet in 500 microliters of IB2 Transfer the supernatant to a new 2 milliliter pre-chilled micro centrifuge tube and label it as supernatant number two, or SN2. Re suspend the final mitochondrial pellet in 200 microliters of re suspension buffer.Quickly set aside 10 microliters for protein quantification and immediately add 10 microliters of 10%FFA BSA to the remaining mitochondrial suspension to prevent damage. Centrifuge the concentrated mitochondrial suspension at 10, 500 times G for 10 minutes at four degrees Celsius. Re suspend the mitochondrial pellet in 100 microliters of 1X coupling assay medium with BSA, 1X electron flow assay medium with BSA, or 1X beta oxidation medium with BSA, depending on the protocol to be performed.Dilute this concentrated mitochondrial sample to 0.2 micrograms per microliter using the corresponding assay medium. Seed 50 microliters of the suspension per well in a pre-chilled 24 well micro plate on ice. In the background correction wells, add the corresponding assay medium only.Store the remaining mitochondrial suspension at minus 80 degrees Celsius for fragmentation purity determination. Spin the micro plate in the pre chilled preparative centrifuge for 20 minutes. In the meantime, warm the remaining assay medium to 37 degrees Celsius.After centrifugation, leave the micro plate on the bench for five minutes to equilibrate. Add 450 microliters of the warm assay medium per well slowly and carefully to avoid detaching of mitochondria. Place the micro plate immediately in the oxygen consumption measuring instrument without the lid and start the measurement program.Ensure that the first step is a 10 minute incubation to allow the micro plate to warm up. Once the experiment is finished, remove the cartridge and micro plate, switch off the instrument, and start the analysis. General protein profiles from the different fractions obtained through serial centrifugations showed distinct protein populations in the isolated fractions.The presence of all the mitochondrial content is evident by the strong signals for markers of the outer and inner mitochondrial membrane and nucleoid associated proteins. Fraction N represents nuclei and undisrupted material. Most of the endoplasmic reticulum, plasma membrane, or cytoplasm markers in the SN1 or SN2 fractions highlight the high purity obtained during isolation.An increase in oxygen consumption was observed after ADP addition in the coupling and beta oxidation assays. After carbonyl cyanide-p-trifluoromethoxy phenylhydrazone addition, oxygen consumption reached its highest level. A low respiratory control ratio ensures the integrity of the isolated mitochondria.The electron flow assay examined the activity of the electron transport chain complexes individually or in combination through the injection of specific substrates and inhibitors. Isolating mitochondria are extremely sensitive. Hence all the steps after tissue mitigation need to be performed diligently and carefully to preserve their viability.Experimental assays can be complimented with a deterministic sematic analysis of key mitochondria molecules or with the study of the assembly of respirometric complexes using blue native polyacrylamide gel electrophoresis.

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4.7K visualizações.  Universidad Pablo de Olavide. Esta palavra descreve a análise de respiração no processamento de mitocôndrias do músculo esquelético do rato. Este protocolo reduz consideravelmente o tempo de purificação e permite que várias amostras sejam processadas simultaneamente. Mitocôndrias de animais de qualquer oito sexo de fundo united podem ser mutiladas usando este protocolo permitindo o estudo da respiração em uma ampla variedade de condições experimentais.Este método é adequado para todas as áreas de p...