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

Les études animales ont été effectuées en vertu d&#39;un protocole approuvé par le Institutional Animal Care et utilisent Comité à Virginia Polytechnic Institute et State University. 1. Configuration (Durée: ~ 45 min) <ol><li> Décongeler les magasins de 0,25% de trypsine, Isolation tampon pour mitochondries (IBM) 1 et IBM2 dans un bain d&#39;eau à 37 ° C. </li><li> Rincer la verrerie et instruments de dissection éthanol à 70% puis à l&#39;eau de haute pureté. </li><li> Préparer la solution de trypsine à 0,05% de 0,25% de trypsine de stock en diluant 1 partie trypsine en 4 parties IBM1. </li><li> Mélanger la protéase et inhibiteur de phosphatase cocktail avec un tampon de lyse cellulaire (1: 100 du rapport) dans un tube de 1,5 ml avec bouchon à vis. </li><li> Aliquotes de 5 ml (5 ml / souris) de 0,05% de trypsine dans un tube en plastique de 15 ml. </li><li> Réglez motorisé homogénéisateur de tissu à 80 RPM. </li></ol> 2. Isolement du muscle squelettique mitochondries (Durée: ~ 90 min) <ol><li> Euthanasier une souris par le CO 2 </sub&gt; inhalation, suivie par dislocation cervicale. </li><li> Retirer muscle rouge du quadriceps et le muscle jumeau, qui comprend le soléaire (~ 75-100 mg au total) comme décrit dans les étapes suivantes: <ol><li> Peler la peau vers la souris. </li><li> Retirer le coussinet adipeux sur le point d&#39;origine quadriceps. Couper le tendon du quadriceps qui est attaché à la rotule avec des ciseaux fins à bout. Remarque: le quadriceps est identifié par sa position anatomique sur la partie antérieure du fémur proximal. </li><li> Lentement couper l&#39;aponévrose entre l&#39;os et les quadriceps, tout en évitant l&#39;artère fémorale, pour libérer les quadriceps de l&#39;os. Couper le tendon avec des ciseaux fins à bout au point d&#39;origine sur le fémur pour libérer le muscle quadriceps et placer le muscle quadriceps en réfrigérés PBS. </li><li> Retirer le tissu adipeux visible sur les quadriceps avec des ciseaux. Retourner les quadriceps plus de sorte que la partie du muscle qui a été recouvrant le fémur est confronté à up. Ouvrez le muscle quadriceps avec une pince dans un mouvement de Fanning. Retirez les deux parties visibles musculaires rouges de chaque lobe avec des ciseaux pointe fine et les placer dans un bécher contenant 5 ml de IBM1 réfrigérés. Remarque: muscle quadriceps apparaissent comme deux lobes avec une bande de muscle rouge situé près du bord latéral de chaque lobe. </li><li> Couper la peau recouvrant le tendon d&#39;Achille avec de fines pointe des ciseaux et peler la peau vers la souris. Couper le tendon d&#39;Achille exposé et peler le muscle vers le corps de la souris. </li><li> Couper le tendon à la condyles latéral et médial du fémur avec des ciseaux pointe fine pour libérer les jumeaux et placer les jumeaux attachée à ses tendons en réfrigérés PBS. </li><li> Retournez le jumeau plus et de décoller le muscle soléaire et le placer dans le bol contenant 5 ml de IBM1 réfrigérés. Fan ouvrir le jumeau. Retirez les trois parties visuelles rouges musculaires (deux bandes rouges latérales et médiale superficielles) avec fine embout ciseauxs et de les transférer dans le bécher contenant 5 ml de IBM1 réfrigérés. </li></ol></li><li> Retirer autre ~ 75 à 100 mg de muscle rouge à partir de l&#39;autre branche de la souris (comme décrit à l&#39;étape 2.2) et la placer dans un tube de 1,5 ml avec une protéase et un inhibiteur de la phosphatase cocktail et un tampon de lyse cellulaire (50 mM Tris-HCL, 1 mM EDTA, 150 mM de NaCl, 1% de dodécylsulfate de sodium, 0,5% de désoxycholate de sodium, 1% de polyoxyéthylène (9) nonylphényléther, ramifiée. Immédiatement Flash geler ce deuxième échantillon dans de l&#39;azote liquide pour une utilisation ultérieure dans le Western blot (voir l&#39;étape 6.1). </li><li> Placez une surface en plastique pré-réfrigérés, à plat sur la glace dans un grand seau. Versez une goutte d&#39;IBM1 sur la surface en plastique et utiliser des pinces pour placer la totalité du muscle rouge coupe (étape 2.2.4 et 2.2.7) dans IBM1 gouttelettes. Émincer le tissu musculaire pendant 2 minutes en utilisant 3 simples lames de rasoir bord en changeant tous les rasoirs 40 sec. </li><li> Transférer le tissu haché à un nouveau bécher avec 5 ml de IBM1 frais en maintenant la surface de plastique sur ee bêcher et racler le muscle dans le bécher avec une lame de rasoir. Prenez cette solution et les égoutter dans une passoire cellulaire 100 um placé sur un tube conique de 50 ml. </li><li> Tamponnez le tissu avec une tâche délicate glace, puis le transférer dans 5 ml de la solution de trypsine à 0,05%. Utilisez une pince à épiler pour enlever le tissu de la tâche essuie-glace. </li><li> Incuber le tissu musculaire sur de la glace pendant 30 min dans une solution de trypsine à 0,05%. </li><li> Faites tourner les 15 ml tube conique mélange contenant de la trypsine / musculaire à 200 g pendant 3 min à 4 ° C. </li><li> Verser le surnageant de la trypsine dans un conteneur de déchets et resuspendre le culot avec 3 ml de glace IBM1 froid. Transférer le tissu dans un tube de verre de 45 homogénéisateur ml. Rincer les 15 ml tube conique avec un autre 1,5 ml IBM1 de rassembler tout échantillon restant et ajouter ce au tube d&#39;homogénéisation de verre. </li><li> Placer le tube de homogénéiseur en verre dans un bécher ou récipient en plastique à mi-chemin rempli de glace afin d&#39;homogénéisation de verre se déplace peu dans le bécher. </li><li&gt; Homogénéiser le mélange tissu / IMB1 avec le pilon en PTFE attachés à une homogénéisation de tissu motorisé avec 10 passes à 80 tours par minute. Maintenir le fond de chaque passage de ~ 2 sec. </li><li> Transférer le homogénat de tissu dans un nouveau tube de 15 ml conique et le tube de rinçage homogénéisateur en verre avec 6,5 ml de IBM1. Verser le IBM1 dans le tube conique contenant 15 ml de l&#39;homogénat tissulaire. </li><li> Faites tourner la 15 ml tube conique à 700 g pendant 10 min à 4 ° C. Verser doucement le surnageant dans un haute résistance tube de centrifugeuse en verre et jeter le culot. </li><li> Spin le surnageant de l&#39;étape précédente à 8000 g pendant 10 min à 4 ° C. </li><li> Eliminer le surnageant IBM1 et remettre en suspension le culot par addition lente de 500 ul de IBM2. Coupez l&#39;extrémité d&#39;une pointe de pipette et homogénéiser doucement le culot dans IBM2 avec mélangeant et en agitant des motions. Ajouter encore 4,5 ml de IBM2 au mélange après que le culot est entièrement suspendu. Remarque: Evitez de trituration excessive tout en brisant les gros morceaux de boulettes que tson peut endommager les membranes mitochondriales. </li><li> Spin le broyat / tissu IBM2 à 8000 g pendant 10 min à 4 ° C. Remarque: Cette étape peut être répétée dans un tube de centrifugeuse à haute résistance propre pour la quantification plus précise de la protéine de mitochondries isolées à l&#39;étape 4 depuis BSA résiduelle peut contribuer à teneur totale en protéines. </li><li> Retirer le surnageant et doucement, mais complètement re-suspendre le culot en ajoutant deux paliers de 25 pi de IBM2 avec une pointe de pipette avec son point coupé. Incorporer délicatement le culot et mélanger après chaque addition de 25 pl de IBM2. Placez ce stock mitochondrial sur la glace. </li></ol> 3. Homogénéisation de tout le tissu Lysate (Durée: ~ 45 min; Peut être effectué pendant l&#39;étape 7) <ol><li> Enlever le muscle de celle a été placé dans de l&#39;azote liquide provenant de l&#39;étape 2.3 et incuber à température ambiante jusqu&#39;à ce que complètement décongelé. </li><li> Émincer le tissu pour ~ 30 secondes sur la glace avec des ciseaux fins à bout. </li><li> Ajouter deux cuillères 100 pi de Zirconium oxyde Perles au tube de 1,5 ml avec bouchon à vis de l&#39;étape 3.2 qui contient le tissu haché. Homogénéiser le tissu dans un homogénéisateur de tissu à l&#39;aide broyeur à billes de deux à trois cycles de 5 min à une vitesse de 4-6 (réglage moyen). </li><li> Spin mélange de l&#39;étape 3.3 à 12000 g pendant 10 min à 4 ° C. Retirer le surnageant à l&#39;aide d&#39;une pipette de 1000 pi après le spin et le transfert dans un tube de 1,5 ml. Jeter le culot et placer le surnageant sur de la glace. </li></ol> 4. Détermination des protéines (Durée: ~ 30 min) <ol><li> Déterminer la concentration en protéine de l&#39;ensemble du lysat de tissu (étape 3.4) et clips mitochondrial (étape 2.17) en utilisant le kit de dosage de protéine BCA de selon les spécifications du fabricant. </li></ol> 5. Les mitochondries Membrane intégrité; Citrate synthase (CS) Activité (Durée: ~ 15 min) <ol><li> Faire deux séparées 1: 20 dilutions du stock mitochondrial dans l&#39;eau de haute pureté. Ajouter0,1% de Triton X-100 à un échantillon et soniquer il à un niveau faible (765 Watts, amplitude de 2). Laissez l&#39;autre dilution sur la glace. Remettre l&#39;échantillon à la glace après sonication. </li><li> Effectuez un test de la citrate synthase à la fois la non-traité par ultrasons et soniqué dilutions mitochondriales en utilisant un dosage spectrophotométrique comme décrit précédemment 11,12. </li><li> Déterminer le pourcentage de membranes intactes mitochondries en utilisant les équations suivantes: (CS activité non-traitées aux ultrasons ÷ soniqué activité CS) * 100 </li></ol> 100- N (pour cent atteint à partir de ci-dessus) 6. Les mitochondries qualité d&#39;isolation; Western Blot (Durée: Conformément au protocole Lab) <ol><li> Effectuer Western blot sur ​​l&#39;ensemble du lysat de tissu et de mitochondries isolées comme décrit précédemment 12. Remarque: Utilisez anticorps primaires pour GAPDH (1: 1000 dilution dans 5% de BSA / TBS-T) et COXIV (1: 1000 dilution dans 5% non graslait / TBS-T), suivie par anti-chèvre, et des anticorps secondaires de lapin, respectivement (1: 10,000). </li></ol> 7. respirométrique série de test (Durée: ~ 90 min) <ol><li> Effectuer des dosages respirométriques souhaités en utilisant la technologie de mesure de microplaques basé O 2 de consommation comme décrit précédemment (voir l&#39;article de compagnon et Rogers et al 8). </li><li> Déterminer le rapport de contrôle respiratoire (RCR) en divisant par 3 État État 4o (RCR) ou en divisant 3U d&#39;État par État 4o. Utilisez soit le milieu (moyenne) le point, ou le plus haut (Etat 3) et les plus faibles (de taux 4o de l&#39;Etat) à partir des traces de point à point au moment de déterminer RCR. </li></ol>

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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

Les méthodes présentées ici fournissent une description détaillée de la procédure d&#39;isolement des mitochondries à partir de quantités minimes (~ 75 à 100 mg) de muscle squelettique de souris. Cette procédure d&#39;isolement est capable de produire de haute fonctionnement, les mitochondries pur (~ 450 ug) comme en témoigne O 2 taux de consommation, les valeurs RCR, l&#39;activité de la citrate synthase maximale et l&#39;expression de protéines à partir immunoblotting. Fait important, les mito...

The primary function of mitochondria is to produce ATP from oxidative phosphorylation. However, mitochondria have many other important cellular functions including but not limited to: the production and detoxification of reactive oxygen species, the regulation of cytoplasmic and mitochondrial calcium, organelle trafficking, ionic homeostasis, and involvement in apoptosis1,2. Therefore, it is not surprising that dysfunctional mitochondria play a role in many disease pathologies, such as aging, neurodegenerative diseases, cardiovascular disease, cancer, obesity, and diabetes3,4. Importantly, skeletal muscle mitochondria specifically are involved in many of these pathologies3-5.
Mitochondrial respiration assays using isolated mitochondria allow for the assessment of electron transport chain and oxidative phosphorylation function, and the determination of mechanism(s) of action of drugs and proteins that modulate metabolism. Mitochondrial isolation procedures exist for multiple tissue and cell types for a variety of species6,7. However, these procedures often require large quantities of tissue/cells for a high quality mitochondria yield necessary for classic respirometric assays. 
Microplate based respirometirc assays allow for high throughput measurements using minimal quantities of isolated mitochondria, often just several &#181;g per well8. Therefore, we present a modification of previously published methods7 to allow for high quality mitochondria to be isolated from smaller quantities of mouse skeletal muscle for use in microplate based respirometirc assays. In addition, methods are provided to establish the quality of the mitochondrial isolation preparation and the integrity of the mitochondrial membranes. Given that skeletal muscle mitochondria are involved in many pathological conditions, the measurement of O2 consumption in mechanistically driven studies is becoming more prevalent in biomedical research9,10.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Essentially Fatty</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A6003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
			<td>Acid Free- BSA</td>
		</tr>
		<tr>
			<td>Tris/HCl</td>
			<td>Promega</td>
			<td>H5123</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Promega</td>
			<td>H5135</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E6511</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>D-Mannitol</td>
			<td>Sigma Aldrich</td>
			<td>63559</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200-056</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium Chloride 
			White Crystals or Crystalline Powder 
			&#8805;99.0 %</td>
			<td>Fisher Scientific</td>
			<td>BP3581</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma Aldrich</td>
			<td>L3771&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma Aldrich</td>
			<td>D6750&#160;</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Polyoxyethylene (12) nonylphenyl ether, branched</td>
			<td>Sigma Aldrich</td>
			<td>238651</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Single Edge Razor Blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Falcon- 100 uM Nylon Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>352360</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td rowspan="2">Halt Protease &#38; Phosphatse Inhibitor Cocktail</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">1861284</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">1.5mL microcentrifuge tubes with screw cap</td>
			<td rowspan="2">Thermo Scientific</td>
			<td rowspan="2">3474</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Zirconium Oxide beads</td>
			<td rowspan="2">Fisher Scientific</td>
			<td rowspan="2">C9012112</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>GAPDH antibody (1D4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-59540</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Anti- COXIV antibody</td>
			<td>Cell Signaling</td>
			<td>4844s</td>
			<td>Any mitochondrial inner membrane protein will suffice</td>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Donkey, Anti Rabbit IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">711-035-152</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Peroxidase conjugated affinipure Goat, Anti Mouse IgG (H+L)</td>
			<td rowspan="2">Jackson ImmunoResearh</td>
			<td rowspan="2">115-001-003</td>
			<td rowspan="2">N/A</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Pyruvic Acid, 98%</td>
			<td>Sigma Aldrich</td>
			<td>107360</td>
			<td>Store at 4&#176;C,pH to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td>Succinic Acid</td>
			<td>Sigma Aldrich</td>
			<td>S9512</td>
			<td>Store at room temperature, pH to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td>L(-) Malic Acid, BioXtra, &#8805;95%</td>
			<td>Sigma Aldrich</td>
			<td>M6413</td>
			<td>Store at room temperature, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
			<td rowspan="2">L-Glutamic acid</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">G1251</td>
			<td rowspan="2">Store at room temperature, to 7.4 with KOH prior to use in respirometric assay, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Palmitoyl L-carnitine chloride</td>
			<td>Sigma Aldrich</td>
			<td>P1645</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Oligomycin A, &#8805; 95% (HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>75351</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)</td>
			<td rowspan="3">Sigma Aldrich</td>
			<td rowspan="3">C2920</td>
			<td rowspan="3">Store at 2-8&#176;C</td>
		</tr>
		<tr>
			<td>phenylhydrazone</td>
		</tr>
		<tr>
			<td>&#8805;98% (TLC), powder [FCCP]</td>
		</tr>
		<tr>
			<td>Antimycin A from streptomyces sp.</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">Adenosine 5&#8242;-diphosphate monopotassium salt dehydrate [ADP]</td>
			<td rowspan="2">Sigma Aldrich</td>
			<td rowspan="2">A5285</td>
			<td rowspan="2">Store at -20&#176;C, to 7.4 with KOH prior to use in respirometric assay</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma Aldrich</td>
			<td>R8875</td>
			<td>Store at room temperature</td>
		</tr>
	</tbody>
</table>

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

Le dysfonctionnement des mitochondries des muscles squelettiques jouent un rôle dans le métabolisme est modifié observée avec le vieillissement, l&#39;obésité et le diabète de type II. Des dosages de préparations mitochondriales respirométriques mitochondries isolées permettent l&#39;évaluation de la fonction mitochondriale, ainsi que la détermination du mécanisme (s) d&#39;action des médicaments et des protéines qui modulent le métabolisme. Procédures d&#39;isolement actuelles exigent souvent de grandes quantités de tissu pour donner mitochondries de haute qualité nécessaires pour les essais respirométriques. Les méthodes présentées ici décrivent comment haute qualité mitochondries purifiées (~ 450 ug) peut être isolé à partir de quantités minimes (~ 75-100 mg) de muscle squelettique de souris pour une utilisation dans des mesures respiratoires à haut débit. Nous avons déterminé que notre méthode d&#39;isolement donne 92,5 ± 2,0% mitochondries intactes en mesurant l&#39;activité de la citrate synthase spectrophotométrie. En outre, l&#39;analyse Western blot dans les mitochondries isolées abouti à l&#39;expression faibles de l&#39;cytosoprotéines lic, GAPDH, et l&#39;expression robuste de la protéine mitochondriale, COXIV. L&#39;absence d&#39;une bande de GAPDH de premier plan dans les mitochondries isolées est indicatif de la contamination à partir de sources peu de non-mitochondriales au cours de la procédure d&#39;isolement. Plus important encore, la mesure de O 2 le taux de consommation sur la base d&#39;une technologie de micro-plaque et la détermination du rapport de contrôle respiratoire (RCR) pour des essais couplés respirométriques montre très couplés (RCR;&gt; 6 pour tous les essais) et les mitochondries fonctionnelles. En conclusion, l&#39;addition d&#39;une étape de hachage séparé et réduisant de manière significative entraîné par un moteur vitesse d&#39;homogénéisation d&#39;un procédé précédemment rapporté a permis l&#39;isolement de haute qualité et des mitochondries purifiées à partir de petites quantités de muscle squelettique de souris qui en résulte dans les mitochondries fortement couplés qui respirent avec une haute fonction lors de microplaques base des essais de respirometirc.

Activité de la citrate synthase sert de mesure pour l&#39;intégrité de la membrane depuis la citrate synthase est situé dans la membrane mitochondriale interne, et par conséquent ne devrait pas être présent dans les suspensions de mitochondries avec des membranes intactes. La figure 1 représente l&#39;activité de la citrate synthase dans des échantillons non soumis à une sonication mitochondriales par rapport à soniqué des échantillons provenant de la même isolation. Soni...

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

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

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

Isolement des mitochondries à partir des quantités minimes de souris muscle squelettique pour les mesures à haut débit microplaques respiratoires

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

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12.7K vues.  Virginia Tech. 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.

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

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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Biologie

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.

Isolement mitochondriale du muscle squelettique

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

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purification des progéniteurs du muscle squelettique

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolement des vaisseaux sanguins dérivés multipotentes précurseurs du muscle squelettique humain

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Haut-débit criblage de modulateurs chimique des gènes post-transcriptionnelle réglementées

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Isolement des mitochondries Intact du muscle squelettique par centrifugation différentielle pour respirométriques mesures haute résolution

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolement et caractérisation d’exosomes à partir de fibroblastes musculaires squelettiques

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolement de myocytes auriculaires et ventriculaires murins de haute qualité pour des mesures simultanées des transitoires Ca2+ et du courant calcique de type L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

Préparation d’homogénats de muscles squelettiques de rat pour les mesures de nitrates et de nitrites

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...