Diese Arbeit wurde durch ein Stipendium der National Eye Institute (R01 EY12998) an der FH Andrade unterstützt.

1. Herstellung der Puffer <ol><li> Schalten Sie Zentrifuge 5804R und auf 4 ° C. Schalten Sie Isotemp 3006D Wasserbad und auf 37 ° C. </li><li> Stellen Sie folgende Lösungen vor Muskel-Isolation: <ul><li> PBS: Lösen Phosphat-gepufferter Kochsalzlösung (PBS)-Tabletten in destilliertem Wasser (5 Tabletten / Liter). Gut mischen. </li><li> PBS plus 10 mM EDTA: So bereiten Sie eine 100 ml-Lösung, 2 ml 500 mM EDTA und 98 ml PBS. </li><li> 8X Mitochondrien Puffer: 10,28 g Saccharose für eine Endkonzentration von 0,6 M, 400 mg der freien Fettsäuren Rinderserumalbumin (BSA) für eine Endkonzentration von 0,8%, 2,08 g HEPES für eine Endkonzentration von 160 mM, pH auf 7,4 und QS auf 50 ml mit destilliertem Wasser. </li><li> Isolation Buffer 1 (IB1): In 200 ul 500 mM EDTA zu einer Endkonzentration von 10 mM, 0,392 g D-Mannitol zu einer Endkonzentration von 215 mM, 1,25 ml 8X Mitochondrien-Puffer, pH auf 7,4 und QS bis 10 ml mit destilliertem Wasser. </li><li> Isolation Buffer 2 (IB2): In 60 ul 500 mM EGTA für eine Endkonzentration von 3 mM, 0,392 g D-Mannitol zu einer Endkonzentration von 215 mM, 1,25 ml 8X Mitochondrien-Puffer, pH auf 7,4 und QS bis 10 ml mit destilliertem Wasser. </li><li> Experimentelle Buffer (EB): 100 l von 500 mM MgCl 2 für eine Endkonzentration von 5 mM, 0,392 g D-Mannitol zu einer Endkonzentration von 215 mM, 25 ul von 2,5 mM KH 2 PO 4 für eine endgültige Konzentration von 6,25 pM, 2 ul 100 mM EGTA für eine Endkonzentration von 20 uM, 1,25 ml der mitochondrialen Puffer, pH auf 7,4 und QS bis 10 ml mit destilliertem Wasser. </li></ul></li></ol> 2. Muscle Isolation <ol><li> In einem Eiskübel, legte drei 10 ml-Becher, Potter-Elvehjem Gewebe Mühlen und alle anderen notwendigen Instrumente / supplies auf Eis. Alles muss bleiben eiskalt während des gesamten Experiments. </li><li> Legen Sie IB1 und IB2 in Eiskübel und EB in warmen Wasserbad, wenn Sie fertig. </li><li> In den drei 10 ml Becher, sollten die folgenden Lösungen gegeben werden: <ul><li> Beaker 1: 10 ml PBS </li><li> Beaker 2: 10 ml PBS / EDTA </li><li> Beaker 3: 3 ml IB1 </li></ul></li><li> Tötet eine Ratte menschlich wie von Ihrem lokalen Institutional Animal Care und Verwenden Committee genehmigt. </li><li> Schnell isolieren 250-500 mg der Skelettmuskulatur, spülen Sie im Becherglas 1, dann Becherglas 2 übertragen. </li></ol> 3. Homogenisierung / Mitochondrial Isolation <ol><li> Transfer-Muskel Beaker 3 und fein hacken den Muskel mit einer Schere. </li><li> Übertragen Sie die Lösung für die Potter-Elvehjem Homogenisator. Homogenisieren der Muskel mit einem motorisierten Stößel (einer Bohrmaschine wird die Arbeit zu tun) 10-mal halten den Schlauch in Eis zu allen Zeiten. </li><li> Transfer-Homogenat zu vorgekühlte 2 ml Reaktionsgefäße und Zentrifuge bei 700 g für 10 Minuten bei 4 ° C. </li><li> Überstand in ein neues vorgekühlte Mikrozentrifugenröhrchen. Discard Pellet. </li><li> Centrifuge Überstand bei 10.500 g für 10 Minuten bei 4 ° C. </li><li> Überstand in ein neues vorgekühlte Mikrozentrifugenröhrchen und Label SN1 (Überstand Nummer 1) und Muskel-Typ. </li><li> Re-suspend Pellet in 500 ul IB2. </li><li> Zentrifuge bei 10.500 g für 10 Minuten bei 4 ° C. </li><li> Überstand in ein neues vorgekühlte Mikrozentrifugenröhrchen und Label SN2 (Überstand Nummer 2) und Muskel-Typ. </li><li> Suspend letzten mitochondriale Pellet in 100 ul IB2. </li><li> Re-Spin-Finale mitochondrialen Suspension in Minifuge für ein paar Sekunden. Wenn es eine Pellet-, Transfer Überstand in ein neues vorgekühlte Mikrozentrifugenröhrchen und entsorgen Sie die Pellets. </li><li> Bestimmen Sie Proteinkonzentration mit der Bradford-Assay 8. </li></ol> 4. Kalibrierung der Elektrode HINWEIS: Vollständige Kalibrierung der Elektrode während der mitochondrialen Isolation. <ol><li> 100 ml destilliertem Wasser auf 250 ml Flasche. Lang kräftig für 20 Minuten, um das Wasser mit Luft-Gas-Gleichgewicht zu bringen. </li><li> Fügen Sie ein paar Tropfen 50% KCl zur Oxygraph Elektrode. </li><li> Legen Sie ein kleines Stück von Blue Rizla Zigarettenpapier auf der Elektrode. </li><li> Legen Sie ein Stück PTFE-Membran auf der Oberseite des Zigarettenpapier. </li><li> Wenden Sie den inneren Ring mit dem Ring-Applikator und platzieren Sie dann den Außenring in die Elektrode Nut. </li><li> Connect Elektroden und montieren den Rest der Ausrüstung: größer Kunststoffring Basis mitochondrialer Kammer und Wasserschläuchen. </li><li> Schalten Sie Oxygraph Boxen und starten Oxygraph Software. </li><li> Fügen Sie die äquilibriert Wasser mitochondrialen Kammer. </li><li> Add Rührstab und schalten Sie auf eine Geschwindigkeit von 60 Jahren. </li><li> Beginnen Sie ein neues Experiment. </li><li> Klicken Sie auf Kalibrieren und dann flüssigen Phase Kalibrierung für Box 1. Ändern Temperatur auf 37 ° C und klicken Sie auf &quot;OK&quot; zweimal. </li><li> Als &quot;Override&quot; auf &quot;OK&quot; klicken, und öffnen Sie die Stickstoff-Tank. </li><li> Platzieren Sie die Spitze verbundenStickstoff-Tank in die Kammer und zu etablieren Null Sauerstoff. Klicken Sie auf &quot;OK&quot;, wenn sie von Schaltern &quot;Override&quot;. </li><li> Saugen Sie Wasser und fügen Sie 500 ul der EB-Puffer zur mitochondrialen Kammern. </li><li> Seal Kammer mit Kolben. </li><li> Bei Verwendung mehrerer Boxen, wiederholen Sie die Schritte 11-15 für die Kalibrierung von Box 2. </li></ol> 5. Mitochondriale Atmung <ol><li> Starten Sie ein neues Experiment, lassen Sie es für ca. 1 Minute laufen, um zu signalisieren stabilisieren. </li><li> Add erforderliche Volumen der mitochondrialen Suspension (Schritt 3,11) für 150 ug in jeder Kammer, markieren Veranstaltung und lassen Sie sie für 1 Minute laufen. </li><li> Add 10 pl warmem 250 mm/125 mm Glutamat / Malat für eine Endkonzentration von 5 mM mM/2.5. Mark und für 1 Minute laufen zu lassen. Dies wird als Zustand 2 definiert. </li><li> Add 7,5 ul 10 mM ADP für eine endgültige Konzentration von 150 uM, Marke, und lassen Sie laufen für 30 Sekunden. Dies wird als State 3 definiert. </li><li> Fügen Sie weitere 7,5 ul 10 mM ADP, Marke, und lassen Sie laufen für 1,5 Minuten. </li><li> Add 0,5 ul kaltem 10 mM Oligomycin für eine endgültige Konzentration von 1 nM, Marke, und lassen Sie laufen für 3 Minuten. Dies ist als staatliche 4 definiert. </li><li> Add 1 ul kalter 0,1 mM FCCP für eine endgültige Konzentration von 0,2 uM, Marke, und lassen Sie laufen für 3 Minuten. Dies ist als staatliche 5 definiert. </li><li> Wenn Sie fertig sind, beenden experimentieren und zu speichern. </li><li> Erwerben Atemfrequenz mittels Oxygraph Software. </li><li> Geben Sie Normierungsfaktor: Wenn Zugabe von 150 pg mitochondriale Protein, dem Normierungsfaktor wird 0,15. </li><li> Berechnen normalisiert Atemfrequenz für die verschiedenen Atmung Staaten. </li><li> Berechnen Sie den Respiratory Control-Ratio (RCR), indem State 3 von State 4. </li></ol> 6. Repräsentative Ergebnisse: <img src="/files/ftp_upload/2452/2452fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Zeigt eine repräsentative Verfolgung von Sauerstoffverbrauch durch Skelettmuskulatur Mitochondrien. Nach der Elektrode Signal stabilisiert ist, wird die Mitochondrien Probe auf die Oxygraph Kammer aufgenommen. State 2 Atmung beginnt mit Zusatz von Glutamat und Malat. Die Zugabe von ADP steigt der Sauerstoffverbrauch, die Definition Zustand 3 Atmung. ATP-Synthase wird durch Zugabe von Oligomycin blockiert, um den Zustand 4 Atmung zu erhalten. Schließlich ist FCCP hinzugefügt, um die mitochondriale Atmung (Zustand 5) zu entkoppeln. Tabelle 1 zeigt repräsentative Sauerstoffverbrauch Preise für die Zustände 2, 3, 4 und 5. Die Atmung kontrollieren Verhältnis (EKV) ist für jedes Experiment berechnet. RCR ≥ 4 wird als Beweis für eine tragfähige Mitochondrien Vorbereitung berücksichtigt. <table border="1"><tbody><tr><td> Staaten </td><td> Normalisierte Preise </td></tr><tr><td> State 2 </td><td> 34,74 </td></tr><tr><td> State 3 </td><td> 153,40 </td></tr><tr><td> State 4 </td><td> 16,49 </td></tr><tr><td> State 5 </td><td> 143,70 </td></tr><tr><td> RCR </td><td> 9,30 </td></tr></tbody></table> Tabelle 1. Mitochondriale Atemfrequenz. Representativeoxygen Verbrauch von Skelettmuskel Mitochondrien. Die Werte sind auf die Höhe der Mitochondrien aufgenommen, um die Kammer normalisiert. Die Atmung kontrollieren Verhältnis (EKV) wird durch Division Zustand 3 durch staatliche 4 bestimmt. Ein RCR ≥ 4 stellt eine tragfähige Mitochondrien Vorbereitung.

<ol>
	<li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat Protoc. 2, 287-295 (2007).</li><li>Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+mitochondria+in+health+and+disease.">Roles of mitochondria in health and disease.</a> Diabetes. 53, S96-S102 (2004).</li><li>Johannsen, D. L., Ravussin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mitochondria+in+health+and+disease.">The role of mitochondria in health and disease.</a> Curr Opin Pharmacol. , (2009).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 65, 1-35 (2001).</li><li>Gamboa, J. L., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+content+and+distribution+changes+specific+to+mouse+diaphragm+after+chronic+normobaric+hypoxia.">Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia.</a> Am J Physiol Regul Integr Comp Physiol. , (2009).</li><li>Patel, S. P., Gamboa, J. L., McMullen, C. A., Rabchevsky, A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+respiratory+capacity+in+extraocular+muscle+mitochondria:+evidence+for+intrinsic+differences+in+mitochondrial+composition+and+function.">Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.</a> Invest Ophthalmol Vis Sci. 50, 180-186 (2009).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

Wir präsentieren ein Protokoll, um lebensfähige Mitochondrien aus Skelettmuskulatur zu isolieren. Wenn Ertrag ist ein Problem, kann das Protokoll durch Inkubation der isolierten Muskel in 5 ml PBS/10mM EDTA/0.01% Trypsin für 30 Minuten in Eis verändert werden. Um sicherzustellen, komplette Muskel-Verdau mit Trypsin, muss der Muskel vollständig zerkleinert werden. Nach dem 30-minütigen Inkubation, muss der PBS/10mM EDTA/0.01% Trypsin-Lösung komplett mit 3 ml der Isolation Puffer 1 (IB1) ersetzt werden. Darüber hi...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>95% CO2 / 5% O2 mix</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2754</td>
<td>&nbsp;</td>
<tr>
<td>Blue Rizla Paper</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>890101</td>
<td>&nbsp;</td>
<tr>
<td>Bradford protein assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
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<td>Compressed nitrogen</td>
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<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9647</td>
<td>&nbsp;</td>
<tr>
<td>Ethlyene-glycol-bis-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E3889</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0728</td>
<td>&nbsp;</td>
<tr>
<td>Free fatty acid bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5889</td>
<td>&nbsp;</td>
<tr>
<td>HEPES sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7006</td>
<td>&nbsp;</td>
<tr>
<td>Isotemp 3006D</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
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<td>Male Sprague Dawley Rats</td>
<td>&nbsp;</td>
<td>Harlan</td>
<td>300-500g</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9138</td>
<td>&nbsp;</td>
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<td>Minifuge</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C1301P</td>
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<td>Sigma</td>
<td>O4876</td>
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<td>Oxygen electrode disc</td>
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<td>S1</td>
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<td>Oxygraph Plus V1.01 Software</td>
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<td>Hansatech</td>
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<td>pH-meter</td>
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<td>Mettler Toledo</td>
<td>1225506149</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8416</td>
<td>&nbsp;</td>
<tr>
<td>Potter-Elvehjem homogenizers</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-414-14A</td>
<td>&nbsp;</td>
<tr>
<td>PTFE (0.0125mm &#215; 25mm) membrane</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S4</td>
<td>&nbsp;</td>
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<td>SKIL 3320 drill press</td>
<td>&nbsp;</td>
<td>Hardware store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
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mitochondrial isolation from skeletal muscle

Mitochondrien sind Organellen Steuerung der Leben und Tod der Zelle. Sie in wichtigen Stoffwechselreaktionen beteiligt, synthetisieren die meisten der ATP, und regeln eine Reihe von Signalkaskaden 2,3. Vergangene und gegenwärtige Forscher haben isolierte Mitochondrien aus Ratten und Mäusen Gewebe wie Leber, Gehirn und Herz 4,5. In den letzten Jahren haben viele Forscher auf die Untersuchung der mitochondrialen Funktion von Skelettmuskeln konzentriert. Hier beschreiben wir eine Methode, die wir erfolgreich für die Isolierung von Mitochondrien aus Skelettmuskeln 6 verwendet. Unser Verfahren erfordert, dass alle Puffer und Reagenzien hergestellt sind frisch und müssen etwa 250-500 mg der Skelettmuskulatur. Wir untersuchten Mitochondrien aus Ratte und Maus gastrocnemius und Zwerchfell isoliert und Ratte Augenmuskeln. Mitochondriale Protein-Konzentration wird mit dem Bradford-Assay gemessen. Es ist wichtig, dass die mitochondriale Proben aufbewahrt eiskalt während der Vorbereitung und funktionelle Studien in relativ kurzer Zeit (~ 1 h) durchgeführt werden kann. Mitochondriale Atmung gemessen mittels Polarographie mit einer Clark-Elektrode (Oxygraph System) bei 37 ° C 7. Die Kalibrierung der Sauerstoffelektrode ist ein wichtiger Schritt in diesem Protokoll und muss täglich durchgeführt werden. Isolierte Mitochondrien (150 ug) sind bis 0,5 ml der experimentellen Puffer (EB) aufgenommen. State 2 Atmung beginnt mit Zusatz von Glutamat (5mm) und Malat (2,5 mM). Dann wird Adenosindiphosphat (ADP) (150 nM) zugegeben, um den Zustand 3 zu beginnen. Oligomycin (1 pM), einer ATPase-Synthase-Blocker, wird verwendet, um den Zustand 4 zu schätzen. Schließlich Carbonyl Cyanid p-[Trifluormethoxy]-phenyl-Hydrazon (FCCP, 0,2 uM) wird zugegeben und 5 measurestate oder abgekoppelt Atmung 6. Die Atmung kontrollieren Verhältnis (EKV), das Verhältnis von Staat 3 bis Zustand 4 wird nach jedem Experiment berechnet. Ein RCR ≥ 4 wird als Beweis für eine tragfähige Mitochondrien Vorbereitung berücksichtigt. Zusammenfassend stellen wir eine Methode zur Isolierung von lebensfähigen Mitochondrien aus Skelettmuskeln, die in biochemischen (zB Enzymaktivität, Immundetektion, Proteomik) und funktionelle Untersuchungen (mitochondriale Atmung) genutzt werden können.

Watch this Scientific Journal Video about Mitochondrial Isolation from Skeletal Muscle at JoVE.com

Mitochondrial Isolation from Skeletal Muscle

Dieses Protokoll beschreibt ein Verfahren zur Atmung von Mitochondrien aus Skelettmuskulatur isoliert zu studieren. Diese Methode wurde von Scorrano angepasst<em&gt; Et al.</em&gt; (2007). Die Mitochondrien isoliert Verfahren erfordert etwa 2 Stunden. Die mitochondriale Atmung kann in ca. 1 Stunde abgeschlossen sein.

Mitochondriale Isolation von der Skelettmuskulatur

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

mitochondrial-isolation-from-skeletal-muscle

Research

JoVE Journal

Biology

28.8K Aufrufe.  University of Kentucky. Dieses Protokoll beschreibt ein Verfahren zur Atmung von Mitochondrien aus Skelettmuskulatur isoliert zu studieren. Diese Methode wurde von Scorrano angepasst Et al. (2007). Die Mitochondrien isoliert Verfahren erfordert etwa 2 Stunden. Die mitochondriale Atmung kann in ca. 1 Stunde abgeschlossen sein.

Serie: Mitochondrial Isolation from Skeletal Muscle

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.

Adulten und embryonalen Skelettmuskel Microexplant Kultur und Isolation der Skelettmuskulatur 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 ...

Forschung

Biologie

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.

Reinigung von Vorläufern aus Skelettmuskeln

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.

Isolation und Kultur der einzelnen Muskelfasern und ihre Satelliten aus adulten Skelettmuskel

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.

Isolierung von Blutgefäß abgeleiteten multi Vorläufer von menschlichen Skelettmuskel

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

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

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

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

Isolierung von Mitochondrien von minimalen Mengen an Maus-Skelettmuskel für die Hochdurchsatz-Mikroplatten-Atemmessungen

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

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.

Isolation of Intact Mitochondria von Skeletal Muscle durch differentielle Zentrifugation für hochauflösende Messungen Respirationstest

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.

Isolierung und Charakterisierung von Exosomen aus Skelettmuskelfibroblasten

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 Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolierung menschlicher ventrikulärer Kardiomyozyten aus Vibratome-Cut Myokardscheiben

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

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

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

Isolierung von Mitochondrien aus der Skelettmuskulatur der Maus für respirometrische Assays

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