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

Gli studi su animali sono stati effettuati nel quadro di un protocollo approvato dalla cura degli animali istituzionale e utilizzano Comitato al Virginia Polytechnic Institute e State University. 1. Setup (Tempo: ~ 45 min) <ol><li> Scongelare depositi congelati di 0,25% tripsina, isolamento tampone per mitocondri (IBM) 1 e IBM2 in un bagno d&#39;acqua a 37 ° C. </li><li> Sciacquare vetreria e strumenti di dissezione in etanolo al 70% e poi con acqua ad elevata purezza. </li><li> Preparare la soluzione tripsina 0,05% dal 0,25% tripsina magazzino diluendo 1 parte di tripsina in 4 parti IBM1. </li><li> Mescolare proteasi e fosfatasi inibitore cocktail con tampone di lisi cellulare (1: 100 ratio) in una provetta da 1,5 ml microcentrifuga con tappo a vite. </li><li> Aliquote 5 ml (5 ml / topo) di 0,05% tripsina in un tubo di plastica da 15 ml. </li><li> Impostare motorizzato omogeneizzatore tessuto a 80 giri. </li></ol> 2. Isolamento del muscolo scheletrico mitocondri (Tempo: ~ 90 min) <ol><li> L&#39;eutanasia di un mouse da CO 2 </sub&gt; inalazione, seguita da dislocazione cervicale. </li><li> Rimuovere muscolo rosso dal quadricipite e del muscolo gastrocnemio, che comprende il soleo (~ 75-100 mg totale) come descritto nei seguenti passaggi: <ol><li> Sbucciare la pelle verso il mouse. </li><li> Rimuovere il cuscinetto di grasso sopra il punto di origine del quadricipite. Tagliare il tendine del quadricipite che è collegato alla rotula con sottili forbici a punta. Nota: Il quadricipite è identificato dalla sua posizione anatomica sulla parte anteriore del femore prossimale. </li><li> Lentamente tagliare l&#39;aponeurosi tra l&#39;osso e il quadricipite, evitando l&#39;arteria femorale, per liberare i quadricipiti dall&#39;osso. Tagliare il tendine con sottili forbici a punta in corrispondenza del punto di origine sul femore per liberare il muscolo quadricipite e posizionare il muscolo quadricipite in refrigerate PBS. </li><li> Rimuovere il tessuto adiposo visibile sopra il quadricipite con le forbici. Vibrazione quadricipiti sopra in modo che la porzione del muscolo che è stato sovrapposto il femore sta affrontando up. Aprire il muscolo quadricipite con una pinza in un movimento rotatorio. Rimuovere le due porzioni muscolari rosse visibili da ogni lobo con ammenda forbici a punta e mettere in un bicchiere contenente 5 ml di acqua refrigerata IBM1. Nota: muscolo quadricipite appaiono come due lobi con una striscia di muscolo rosso si trova vicino al bordo laterale di ciascun lobo. </li><li> Tagliare la pelle sovrastante il tendine di Achille con ammenda punta forbici e sbucciare la pelle verso il mouse. Tagliare il tendine di Achille esposto e staccare il muscolo verso il corpo del mouse. </li><li> Tagliare il tendine ai condili mediale e laterale del femore con sottili forbici a punta per liberare il gastrocnemio e collocare il gastrocnemio attaccato ai suoi tendini in refrigerata PBS. </li><li> Capovolgere il gastrocnemio sopra e staccare il muscolo soleo e mettere nel bicchiere contenente 5 ml di acqua refrigerata IBM1. Fan aprire il gastrocnemio. Rimuovere le tre porzioni visive rosse muscolari (due strisce rosse laterali e uno mediale superficiale) con multa forbice a puntas e trasferirli al bicchiere contenente 5 ml di acqua refrigerata IBM1. </li></ol></li><li> Rimuovere un&#39;altra ~ 75-100 mg di muscolo rosso dalla altra gamba del mouse (come descritto al punto 2.2) e posto in una provetta da 1,5 ml microcentrifuga con proteasi e fosfatasi inibitore cocktail e tampone di lisi cellulare (50 mM Tris-HCL, 1 mM EDTA, NaCl 150 mm, 1% dodecil solfato di sodio, 0,5% deossicolato di sodio, 1% Poliossietilene (9) nonylphenylether, ramificata. Subito lampeggiare-freeze questo secondo campione in azoto liquido per un uso successivo a assorbente occidentale (vedi punto 6.1). </li><li> Mettere una superficie di plastica piatta pre-raffreddata su ghiaccio in un grande secchio. Versare una goccia di IBM1 sulla superficie di plastica e utilizzare pinzette per mettere tutti del muscolo rosso sezionato (passo 2.2.4 e 2.2.7) in IBM1 goccia. Tritare il tessuto muscolare per 2 minuti utilizzando 3 singole lame di rasoio bordo cambiando rasoi ogni 40 sec. </li><li> Trasferire il tessuto tritato in un becher con 5 ml di IBM1 fresco tenendo superficie plastica sopra the becher e raschiare il muscolo nel becher con una lama di rasoio. Prendete questa soluzione e di scarico attraverso un colino cella di 100 micron collocato su un tubo da 50 ml. </li><li> Tamponare il tessuto con un tergicristallo compito delicato e poi trasferisce in 5 ml della soluzione di tripsina 0,05%. Utilizzare pinzette per rimuovere il tessuto dal tergicristallo compito. </li><li> Incubare il tessuto muscolare in ghiaccio per 30 minuti in soluzione tripsina 0,05%. </li><li> Gira le tubo da 15 ml miscela contenente tripsina / muscolo a 200 xg per 3 min a 4 ° C. </li><li> Versare il surnatante tripsina in un contenitore per rifiuti e risospendere il pellet con 3 ml di ghiaccio freddo IBM1. Trasferire il tessuto 45 ml tubo omogeneizzatore vetro. Sciacquare i tubo da 15 ml con un altro 1,5 ml IBM1 raccogliere qualsiasi campione rimanente e aggiungere questo al tubo omogeneizzatore vetro. </li><li> Posizionare il tubo di omogeneizzatore vetro in un bicchiere o contenitore di plastica pieno di ghiaccio a metà strada quindi l&#39;omogeneizzatore vetro si muove minimamente all&#39;interno del bicchiere. </li><li&gt; Omogeneizzare la miscela di tessuti / IMB1 con il pestello in PTFE collegati ad un comando a motore omogeneizzatore tessuto con 10 passaggi a 80 giri al minuto. Tenere la parte inferiore di ogni pass per ~ 2 sec. </li><li> Trasferire il tessuto omogenato di un nuovo tubo da 15 ml conica e sciacquare il tubo omogeneizzatore vetro con 6,5 ml di IBM1. Versare il IBM1 nel tubo da 15 ml contenente tessuto omogenato. </li><li> Spin tubo conico da 15 ml a 700 g per 10 minuti a 4 ° C. Delicatamente versare il surnatante in una provetta da centrifuga ad alta resistenza vetro e scartare il pellet. </li><li> Spin il supernatante dal passo a 8000 xg per 10 min a 4 ° C. </li><li> Rimuovere il surnatante IBM1 e risospendere il pellet aggiungendo lentamente 500 ml di IBM2. Tagliare la fine di un puntale e omogeneizzare delicatamente il pellet in IBM2 con miscelazione e agitazione movimenti. Aggiungere altri 4,5 ml di IBM2 alla miscela dopo il pellet è completamente sospesa. Nota: Evitare l&#39;eccessiva triturazione mentre rompendo pezzi pellet più grandi come tla sua può danneggiare le membrane mitocondriali. </li><li> Spin l&#39;omogeneizzato / tessuto IBM2 a 8000 xg per 10 minuti a 4 ° C. Nota: Questo passaggio può essere ripetuto in una provetta pulita centrifuga ad alta resistenza per più precisa quantificazione delle proteine ​​di mitocondri isolati al punto 4 dal residuo BSA può contribuire al contenuto totale di proteine. </li><li> Rimuovere il surnatante e delicatamente, ma completamente risospendere il pellet con l&#39;aggiunta di due incrementi 25 microlitri di IBM2 con un puntale con il suo punto di taglio. Mescolare delicatamente e miscelare il pellet dopo ogni aggiunta di 25 ml di IBM2. Mettere questo stock mitocondriale su ghiaccio. </li></ol> 3. omogeneizzazione di tutto il tessuto lisato (Tempo: ~ 45 min; può essere effettuato durante Passo 7) <ol><li> Rimuovere il muscolo da che è stato posto in azoto liquido dal punto 2.3 e incubare a temperatura ambiente fino completamente scongelati. </li><li> Tritare il tessuto per ~ 30 secondi sul ghiaccio con pregiati forbici a punta. </li><li> Aggiungere due cucchiai 100 microlitri di ZircoPerline in ossido di zirconio alla provetta da 1,5 ml microcentrifuga con tappo a vite dal punto 3.2 che contiene il tessuto tritato. Omogeneizzare il tessuto in un omogeneizzatore tessuto mulino tallone con due o tre cicli di 5 min ad una impostazione di velocità di 4-6 (regolazione media). </li><li> Spin la miscela dal punto 3.3 a 12.000 xg per 10 min a 4 ° C. Rimuovere il surnatante con una pipetta 1000 ml punta dopo lo spin e il trasferimento in una provetta da 1,5 ml microcentrifuga. Eliminare il pellet e posizionare il surnatante su ghiaccio. </li></ol> 4. Determinazione delle proteine ​​(Tempo: ~ 30 min) <ol><li> Determinare la concentrazione di proteine ​​di tutto il lisato tissutale (punto 3.4) e magazzino mitocondriale (passo 2.17) utilizzando il kit di analisi delle proteine ​​BCA secondo le specifiche del costruttore. </li></ol> 5. I mitocondri Membrana di integrità; Citrato sintasi (CS) Attività (Tempo: ~ 15 min) <ol><li> Fare due separati 1: 20 diluizioni dello stock mitocondriale in acqua ad elevata purezza. Aggiungere0,1% di Triton 100-X per un campione e sonicare è a un livello basso (765 watt, l&#39;ampiezza di 2). Lasciare l&#39;altra soluzione sul ghiaccio. Riportare il campione ghiaccio dopo sonicazione. </li><li> Eseguire un test citrato sintasi sia con il non-sonicato e sonicato diluizioni mitocondriali utilizzando un saggio spettrofotometrico precedentemente descritto 11,12. </li><li> Determinare la percentuale delle membrane mitocondriali intatte in base alle seguenti equazioni: (Attività CS non sonicato ÷ sonicato CS attività) * 100 </li></ol> 100- N (cento raggiunto da sopra) 6. I mitocondri isolamento di qualità; Western Blotting (Tempo: Secondo Lab Protocol) <ol><li> Eseguire Western blotting su tutta lisato tissutale e mitocondri isolati come descritto in precedenza 12. Nota: Utilizzare anticorpi primari per GAPDH (1: 1.000 diluizione 5% BSA / TBS-T) e COXIV (1: 1.000 diluizione 5% senza grassiLatte / TBS-T), seguita da anti-capra, e anticorpi secondari di coniglio, rispettivamente, (1: 10.000). </li></ol> 7. respirometrici Assay Run (Tempo: ~ 90 min) <ol><li> Effettuare test respirometrici desiderati usando basato O 2 tecnologia di misurazione del consumo di micropiastre come descritto in precedenza (vedi articolo correlato e Rogers et al 8). </li><li> Determinare il rapporto di controllo respiratorio (RCR) dividendo Stato 3 per Stato 4o (RCR) o dividere 3u Stato per Stato 4o. Utilizzare il mezzo (media) il punto, o il più alto (Stato 3) e tariffe più basse (4o Stato) dal punto a punto le tracce per determinare RCR. </li></ol>

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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>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> Biotechnol. and Bioeng. 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> Nat. Protoc. 1, 2563-2572 (2007).</li><li>Hulver, M. 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=Elevated+stearoyl-CoA+desaturase-1+expression+in+skeletal+muscle+contributes+to+abnormal+fatty+acid+partitioning+in+obese+humans.">Elevated stearoyl-CoA desaturase-1 expression in skeletal muscle contributes to abnormal fatty acid partitioning in obese humans.</a> Cell Metab. 2, 251-261 (2005).</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=Toll-like+receptor+4+modulates+skeletal+muscle+substrate+metabolism.">Toll-like receptor 4 modulates skeletal muscle substrate metabolism.</a> Am. J. Physiol. Endocrinol. Metab. 298, e988-e998 (2010).</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> Adv. Enzymol. Relat. Subjects of Biochem. 17, 65-134 (1956).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Populations+of+rat+skeletal+muscle+mitochondria+after+exercise+and+immobilization.">Populations of rat skeletal muscle mitochondria after exercise and immobilization.</a> J. Appl. Physiol.: Respir., Envir. and Ex. Physiol. 48, 23-28 (1980).</li><li>Asmann, Y. 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George Rogers is an employee of Seahorse Bioscience that produces the instrument for which this protocol was modified.

I metodi qui presentati forniscono una descrizione dettagliata di una procedura di isolamento mitocondriale da quantità minime (~ 75-100 mg) di topo muscoli scheletrici. Questa procedura di isolamento è in grado di produrre ad alto funzionamento, i mitocondri puro (~ 450 mg) come evidenziato da O 2 tassi di consumo, valori RCR, la massima attività citrato sintasi e di espressione proteica da immunoblotting. È importante sottolineare che i mitocondri isolati da questa procedura può essere utilizzato per p...

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

Disfunzionali mitocondri dei muscoli scheletrici hanno un ruolo nel metabolismo alterato osservata con l&#39;invecchiamento, l&#39;obesità e il diabete di tipo II. Saggi respirometrici mitocondriali da isolate preparati mitocondriali consentono la valutazione della funzione mitocondriale, nonché la determinazione del meccanismo (s) d&#39;azione dei farmaci e proteine ​​che modulano il metabolismo. Procedure di isolamento attuali richiedono spesso grandi quantità di tessuto per produrre mitocondri di alta qualità necessari per le analisi respirometrici. I metodi qui presentati descrivono come di alta qualità mitocondri purificato (~ 450 mg) può essere isolato da quantità minime (~ 75-100 mg) di topo muscolo scheletrico per l&#39;uso in misure respiratorie throughput elevato. Abbiamo determinato che il nostro metodo di isolamento si ottiene il 92,5 ± 2,0% mitocondri intatti misurando l&#39;attività citrato sintasi spettrofotometricamente. Inoltre, l&#39;analisi Western Blot in mitocondri isolati portato alla debole espressione del cytosoproteine ​​lic, GAPDH, e il robusto espressione della proteina mitocondriale, COXIV. L&#39;assenza di una banda di GAPDH prominente nei mitocondri isolati è indicativo di poco la contaminazione da fonti non-mitocondriali durante la procedura di isolamento. Soprattutto, la misura di O 2 tasso di consumo con tecnologia basata micropiastra e determinare il rapporto di controllo respiratorio (RCR) per saggi respirometrici accoppiati mostra altamente accoppiate (RCR;&gt; 6 per tutti i saggi) e mitocondri funzionali. In conclusione, l&#39;aggiunta di una fase di macinazione separata e riducendo significativamente motorizzato velocità omogeneizzazione di un metodo riportato in precedenza ha permesso l&#39;isolamento di alta qualità e mitocondri purificati da piccole quantità di topo muscoli scheletrici che si traduce nei mitocondri altamente accoppiate che respirano con alta funzione durante micropiastra saggi respirometirc.

Attività citrato sintasi serve come misura per l&#39;integrità della membrana dal citrato sintasi si trova nella membrana mitocondriale interna, e quindi non dovrebbero essere presenti nelle sospensioni di mitocondri con membrane intatte. La Figura 1 rappresenta l&#39;attività citrato sintasi in campioni non sonicato mitocondriali rispetto sonicato campioni dello stesso isolamento. Sonicating i risultati dei mitocondri in un aumento statisticamente significativo nell&#39;attività ci...

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.

L&#39;isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

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

isolation-mitochondria-from-minimal-quantities-mouse-skeletal-muscle

Research

JoVE Journal

Biology

12.7K Views.  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.

Video: 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.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

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

Ricerca

Biologia

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.

Isolamento dei mitocondri dal muscolo scheletrico

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.

Purificazione di progenitori dal muscolo scheletrico

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.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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.

L&#39;isolamento di mitocondri intatti dal muscolo scheletrico mediante centrifugazione differenziale per alta risoluzione respirometria Misure

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.

Isolamento e caratterizzazione di esosomi da fibroblasti muscolari scheletrici

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.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo 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.

Preparazione di omogenati muscolari scheletrici di ratto per misurazioni di nitrati e nitriti

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