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

Tierexperimentelle Studien wurden im Rahmen einer genehmigten Protokoll von der Institutional Animal Care und Verwenden Committee durchgeführt am Virginia Polytechnic Institute und State University. 1. Setup (Zeit: ~ 45 min) <ol><li> Tauwetter gefrorenen Filialen von 0,25% Trypsin, Isolation Puffer für Mitochondrien (IBM) 1 und IBM2 in einem 37 ° C Wasserbad. </li><li> Spülen Sie Gläser und Sezieren Instrumente in 70% Ethanol, gefolgt von hochreinem Wasser. </li><li> Bereiten 0,05% Trypsin-Lösung von 0,25% Trypsin Lager durch Verdünnen von 1 Teil Trypsin in 4 Teilen IBM1. </li><li> Mischen Sie Protease und Phosphatase-Inhibitor-Cocktail mit Zelllysepuffer (1: 100-Verhältnis) in einem 1,5 ml Mikrozentrifugenröhrchen mit Schraubverschluss. </li><li> Aliquots von 5 ml (5 ml / Maus) von 0,05% Trypsin in ein 15 ml Kunststoffröhrchen. </li><li> Set Motor angetrieben Gewebehomogenisator zu 80 RPM. </li></ol> 2. Isolierung der Skelettmuskulatur Mitochondrien (Zeit: ~ 90 min) <ol><li> Euthanize eine Maus mit CO 2 </sub&gt; Einatmen, gefolgt durch zervikale Dislokation. </li><li> Entfernen Sie rote Muskel von der Quadrizeps und M. gastrocnemius, der die soleus (~ 75-100 mg insgesamt), wie in den folgenden Schritten beschrieben beinhaltet: <ol><li> Schälen Sie die Haut in Richtung der Maus. </li><li> Entfernen Sie die Fettpolster über der Quadrizeps-Ursprungspunkt. Schneiden Sie die Quadrizeps-Sehne, die die Kniescheibe mit feiner Spitze Schere angebracht ist. Hinweis: Der Quadrizeps durch seine anatomische Position an dem vorderen Abschnitt des proximalen Femur identifiziert. </li><li> Langsam schnippeln die Aponeurose zwischen dem Knochen und der Quadrizeps, unter Vermeidung der Oberschenkelschlagader, um die Quadrizeps aus dem Knochen zu befreien. Schneiden Sie die Sehne mit feiner Spitze Schere am Ursprungspunkt auf dem Femur um den Quadrizeps zu befreien und legen Sie die Quadrizeps in gekühltem PBS. </li><li> Entfernen Sie sichtbaren Fettgewebe in den Quadrizeps mit einer Schere. Flip Quadrizeps um, so dass der Teil des Muskels, der über den Oberschenkelknochen wurde zugewandt ist uSeite Öffnen Sie die Quadrizeps mit einer Pinzette in einem pendelnden Bewegungen. Entfernen Sie die beiden sichtbaren roten Muskelabschnitte von jedem Lappen mit feiner Spitze Schere und in ein Becherglas mit 5 ml gekühlt IBM1. Hinweis: Quadrizeps als zwei Lappen mit einem Streifen aus roten Muskel in der Nähe der Seitenkante jedes Flügels angeordnet angezeigt. </li><li> Schneiden Sie die Haut über der Achillessehne mit feiner Spitze Schere und schälen die Haut in Richtung der Maus. Schneiden Sie die freiliegenden Achillessehne und schälen die Muskeln auf den Körper der Maus. </li><li> Schneiden Sie die Sehne am lateralen und medialen Kondylen des Oberschenkelknochens mit feiner Spitze Schere, um die gastrocnemius zu befreien und legen Sie die gastrocnemius, seine Sehnen in gekühltem PBS angeschlossen. </li><li> Klappen Sie den gastrocnemius auf und ziehen Sie die Soleusmuskel und legen Sie in den Becher, die 5 ml gekühlt IBM1. Fan öffnen Sie die gastrocnemius. Entfernen Sie die drei visuellen roten Muskelabschnitte (zwei seitlichen und einem medialen oberflächliche rote Streifen) mit feinen Spitzen versehenen Scherens und übertragen diese zu dem Becherglas gegeben, 5 ml gekühlter IBM1. </li></ol></li><li> Entfernen weiteres ~ 75-100 mg von roten Muskel aus dem anderen Schenkel des Maus (wie in Schritt 2.2 beschrieben) und in einen 1,5 ml-Mikrozentrifugenröhrchen mit Protease- und Phosphatase-Inhibitor-Cocktail und Zelllyse-Puffer (50 mM Tris-HCL, 1 mM EDTA, 150 mM NaCl, 1% Natriumdodecylsulfat, 0,5% Natriumdeoxycholat, 1% Polyoxyethylen (9) nonylphenylether, verzweigt. Unmittelbar Blitz-einfrieren dieses zweite Probe in flüssigem Stickstoff für die spätere Verwendung in Western Blot (siehe Schritt 6.1). </li><li> Legen Sie eine vorgekühlte, flache Kunststoffoberfläche auf Eis in einem großen Eimer. Gießen Sie einen Tropfen IBM1 auf die Kunststoffoberfläche und verwenden Pinzette, um das gesamte geschnittene rote Muskel (Schritt 2.2.4 und 2.2.7) in IBM1 Tröpfchen zu platzieren. Hackfleisch das Muskelgewebe für 2 min mit 3 Einzelklingen durch Ändern Rasierer alle 40 sec. </li><li> Übertragen des zerkleinerten Gewebes mit einem neuen Becherglases mit 5 ml frischem IBM1, indem die Kunststoffoberfläche, über the-Becherglas und Abkratzen der Muskeln in den Becher mit einer Rasierklinge. Nutzen Sie diese Lösung und lassen es durch einen 100 &amp; mgr; m Zellsieb auf eine 50 ml konischen Röhrchen platziert. </li><li> Tupfen Sie das Gewebe mit einer heiklen Aufgabe Wischer und dann übertragen sie in 5 ml 0,05% Trypsin-Lösung. Verwenden einer Pinzette, um das Gewebe von der Aufgabe Wischer zu entfernen. </li><li> Inkubieren des Muskelgewebes auf Eis für 30 min in 0,05% Trypsin-Lösung. </li><li> Spinnen Sie die 15 ml konischen Röhrchen mit Trypsin / Muskel Mischung bei 200 × g für 3 min bei 4 ° C. </li><li> Gießen Sie die Trypsin-Überstand in einen Abfallbehälter und das Pellet mit 3 ml eiskaltem IBM1. Übertragen Sie das Gewebe bis zu einer 45 ml Glas-Homogenisator Rohr. Mit einem weiteren 1,5 ml IBM1 Spülen Sie die 15 ml konischen Röhrchen, um alle verbleibenden Proben sammeln und fügen Sie diese in die Glas-Homogenisator Rohr. </li><li> Legen Sie das Glas-Homogenisator Rohr in ein Becherglas oder Kunststoffbehälter gefüllt zur Hälfte mit Eis, so dass die Glas-Homogenisator bewegt sich minimal in das Becherglas. </li><li&gt; Homogenisieren des Gewebes / IMB1 Mischung mit den PTFE-Pistill zu einem Motor angetrieben Gewebehomogenisator mit 10 Durchgängen bei 80 UpM angebracht ist. Halten Sie den unteren Rand jeder Pass für ca. 2 Sek. </li><li> Übertragen Sie die Gewebehomogenat auf ein neues 15 ml konischen Rohr und spülen Sie das Glas-Homogenisator Rohr mit 6,5 ml IBM1. Gießen Sie die IBM1 in die 15 ml konischen Röhrchen mit Gewebehomogenat. </li><li> Spin der konischen 15 ml-Röhrchen bei 700 g für 10 min bei 4 ° C. Gießen Sie vorsichtig den Überstand in ein Glas mit hoher Festigkeit Zentrifugenröhrchen und entsorgen Sie das Pellet. </li><li> Spin der Überstand aus dem obigen Schritt bei 8.000 × g für 10 min bei 4 ° C. </li><li> Entfernen Sie den Überstand und IBM1 resuspendieren Sie das Pellet durch langsame Zugabe von 500 ul IBM2. Schnitt das Ende einer Pipettenspitze und schonend homogenisiert das Pellet in IBM2 mit Mischen und Rühren von Bewegungen. Fügen Sie ein weiteres 4,5 ml IBM2 zu der Mischung, nachdem das Pellet vollständig aufgehängt ist. Hinweis: Vermeiden Sie übermäßige Verreiben, während brechen größere Pellet Stücke tsein kann die Mitochondrienmembranen beschädigen. </li><li> Spin der IBM2 / Gewebehomogenisat bei 8.000 × g für 10 min bei 4 ° C. Hinweis: Dieser Schritt kann in einem sauberen hohe Festigkeit Zentrifugenröhrchen für eine genauere Quantifizierung der Protein isolierten Mitochondrien in Schritt 4, da Rest BSA wiederholen kann, um den Gesamtproteingehalt beiträgt. </li><li> Entfernen Sie den Überstand und sanft, aber vollständig resuspendieren Sie das Pellet durch Zugabe von zwei 25 &amp; mgr; l-Schritten IBM2 mit einer Pipettenspitze mit der Spitze abgeschnitten. Vorsichtig umrühren und mischen das Pellet nach jeder Zugabe von 25 ul IBM2. Dieses mitochondriale Lager auf Eis. </li></ol> 3. Homogenisierung Whole Gewebelysat (Zeit: ~ 45 min; kann getan werden während Schritt 7) <ol><li> Entfernen Sie den Muskel aus, dass in den flüssigen Stickstoff aus dem Schritt 2.3 gelegt und bei Raumtemperatur, bis sie vollständig aufgetaut. </li><li> Hackfleisch das Gewebe für ~ 30 sec auf Eis mit feiner Spitze Schere. </li><li> Fügen Sie zwei 100 ul Kugeln Zirconium-Perlen mit dem 1,5-ml-Mikrozentrifugenröhrchen mit Schraubverschluss aus Schritt 3.2, die das zerkleinerte Gewebe enthält. Homogenisieren des Gewebes in einer Perlmühle Gewebehomogenisator mit zwei, um drei 5-Minuten-Zyklen bei einer Geschwindigkeitseinstellung von 4-6 (mittlere Einstellung). </li><li> Spin der Mischung aus Schritt 3.3 bei 12.000 × g für 10 min bei 4 ° C. Entfernen Sie den Überstand mit einer 1.000 ul Pipettenspitze nach dem Spin und Transfer in ein 1,5 ml Mikrozentrifugenröhrchen. Entsorgen Sie das Pellet und platzieren Sie den Überstand auf Eis. </li></ol> 4. Proteinbestimmung (Zeit: ~ 30 min) <ol><li> Bestimmung der Proteinkonzentration des gesamten Gewebelysat (Schritt 3.4) und mitochondriale Lager (Schritt 2.17) unter Verwendung des BCA-Protein-Assay-Kit nach den Angaben des Herstellers. </li></ol> 5. Mitochondrien Membranintegrität; Citratsynthase (CS) Activity (Zeit: ~ 15 min) <ol><li> Machen Sie zwei 1: 20-Verdünnungen der mitochondrialen Lager in hochreinem Wasser. Hinzufügen0,1% Triton X-100 zu einer Probe und beschallen es bei einer niedrigen Einstellung (765 Watts, Amplitude 2). Lassen Sie die anderen Verdünnung auf Eis. Bringen Sie die Probe auf Eis nach der Beschallung. </li><li> Führen Citratsynthase Assay sowohl die nicht-beschallte und beschallt mitochondrialen Verdünnungen mit einem spektrophotometrischen Test, wie zuvor beschrieben 11,12. </li><li> Bestimmen Sie den Prozentsatz der intakten Mitochondrien-Membranen unter Verwendung der folgenden Gleichungen: (Non-beschallt CS Aktivität ÷ beschallt CS-Aktivität) * 100 </li></ol> 100- N (Prozent von oben erreicht) 6. Mitochondrien Isolation Qualität; Western Blot (Zeit: Nach Lab Protocol) <ol><li> Zuführen Western Blot auf die gesamte Gewebelysat und isolierte Mitochondrien, wie zuvor beschrieben 12. Anmerkung: Die Verwendung Primärantikörper für GAPDH (1: 1.000 Verdünnung in 5% BSA / TBS-T) und COXIV (1: 1.000 Verdünnung in 5% fettMilch / TBS-T), gefolgt von anti-Ziege und Kaninchen-Sekundärantikörper, die jeweils (1: 10.000). </li></ol> 7. Respirometrische Testlauf (Zeit: ~ 90 min) <ol><li> Führen Sie gewünschte respirometrischen Tests unter Verwendung von Mikroplatten-basierten O 2 Verbrauchsmesstechnik, wie zuvor beschrieben (siehe Begleitartikel und Rogers et al 8). </li><li> Bestimmen Sie die Atemkontrollverhältnis (RCR) durch Division State 3 durch staatliche 4o (RCR) oder Division Staat 3U durch staatliche 4o. Verwenden Sie entweder die mittlere (durchschnittliche) Punkt oder die höchste (State 3) und die niedrigsten Preise (State 4o) von den Punkt-zu-Punkt-Spuren bei der Ermittlung RCR. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+genome+in+aging+and+senescence.">The mitochondrial genome in aging and senescence.</a> Ageing Res. Rev. 18, 1-15 (2014).</li><li>Dube, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+lipid+overload+on+skeletal+muscle+insulin+resistance,+metabolic+flexibility,+and+mitochondrial+performance.">Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance.</a> Am. J. Physiol. Endocrinol. Metab. 12, 1117-1124 (2014).</li><li>Fernandez-Vizarra, E., Lopez-Perez, M. J., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+biogenetically+competent+mitochondria+from+mammalian+tissues+and+cultured+cells.">Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells.</a> Methods (San Diego, Calif). 26, 292-297 (2002).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat. Protoc. 2, 287-295 (2007).</li><li>Rogers, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microplate+respiratory+measurements+using+minimal+quantities+of+isolated+mitochondria.">High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria.</a> PloS one. 6, e21746 (2011).</li><li>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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> JoVE. , (2011).</li><li>Gross, V. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+functional+mitochondria+from+rat+kidney+and+skeletal+muscle+without+manual+homogenization.">Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization.</a> Anal. Biochem. 418, 213-223 (2011).</li><li>Krieger, D. A., Tate, C. A., McMillin-Wood, J., Booth, F. 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. 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=Skeletal+muscle+mitochondrial+functions,+mitochondrial+DNA+copy+numbers,+and+gene+transcript+profiles+in+type+2+diabetic+and+nondiabetic+subjects+at+equal+levels+of+low+or+high+insulin+and+euglycemia.">Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.</a> Diabetes. 55, 3309-3319 (2006).</li><li>Lanza, I. R., Nair, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+isolated+mitochondria+in+vitro.">Functional assessment of isolated mitochondria in vitro.</a> Methods Enzymol. 457, 349-372 (2009).</li></ol>

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

Die hier vorgestellten Methoden liefern eine detaillierte Beschreibung eines mitochondrialen Isolierungsverfahren von minimalen Mengen (~ 75-100 mg) von Maus-Skelettmuskel. Dieses Isolierungsverfahren ist in der Lage, High-Functioning, reine Mitochondrien (~ 450 &amp; mgr; g) zu ergeben, wie durch O 2 Verbrauchsraten, RCR-Werte, Maximal Citrat-Synthase-Aktivität und Proteinexpression von Immunoblotting nachgewiesen. Wichtig ist, dass die Mitochondrien von diesem Verfahren getrennt für mehrere respirometirc ...

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

Dysfunktionalen Skelettmuskel Mitochondrien spielen eine Rolle in Zusammenhang mit der Alterung, Übergewicht und Diabetes Typ II beobachtet veränderten Stoffwechsel. Mitochondriale respirometrischen Assays aus isolierten Mitochondrien Zubereitungen erlauben die Beurteilung der Mitochondrienfunktion sowie die Bestimmung des Mechanismus (en) der Wirkung von Arzneimitteln und Proteinen, die den Stoffwechsel modulieren. Aktuelle Isolierungsverfahren erfordern häufig große Mengen von Gewebe, um qualitativ hochwertige Mitochondrien für respirometrischen Assays notwendig ergeben. Die hier vorgestellten Methoden beschreiben, wie hohe Qualität gereinigt Mitochondrien (~ 450 &amp; mgr; g) von geringen Mengen (~ 75-100 mg) von Maus-Skelettmuskel isoliert werden für den Einsatz in Hochdurchsatz-Atemmessungen. Wir stellten fest, dass unsere Trennung Verfahren ergibt 92,5 ± 2,0% intakte Mitochondrien durch Messung Citrat-Synthase-Aktivität spektralphotometrisch. Darüber hinaus Western Blot-Analyse in isolierten Mitochondrien führte das schwache Expression des cytosolic Protein, GAPDH und die robuste Expression des mitochondrialen Proteins, COXIV. Die Abwesenheit eines bekannten GAPDH Band in den isolierten Mitochondrien indikativ für kleine Verunreinigungen von nicht mitochondrialen Quellen während des Isolierungsverfahrens. Am wichtigsten ist, die Messung des O 2 Verbrauchsrate mit Mikroplatte basierte Technologie und Bestimmen des respiratorischen Steuerungsverhältnis (RCR) für gekoppelte respirometrischen Assays zeigen hoch gekoppelt (RCR,&gt; 6 für alle Assays) und funktionelle Mitochondrien. Im Ergebnis ist die Zugabe eines separaten Zerkleinern Schritt und Motor angetrieben Homogenisierungsgeschwindigkeit eines zuvor beschriebene Verfahren erheblich reduziert ist die Isolierung von hoher Qualität und gereinigten Mitochondrien aus kleineren Mengen von Maus-Skelettmuskel, in stark gekoppelt Mitochondrien, die mit hoher Funktions respire Ergebnisse führten während auf Mikrotiterplatte respirometirc Assays.

Citrat-Synthase-Aktivität als Maß für die Unversehrtheit der Membran, da Citratsynthase ist in der inneren Mitochondrienmembran, und sollte daher nicht in Suspensionen von Mitochondrien mit intakten Membranen vorliegen. 1 stellt Citrat-Synthase-Aktivität in nicht-beschallten mitochondrialen Proben verglichen, die mit beschallter Proben aus der gleichen Isolation. Beschallen der Mitochondrien führt zu einem statistisch signifikanten Anstieg der Citrat-Synthase-Aktivität (P &lt;0,01). Wichtig ist, d...

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.

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

Research

JoVE Journal

Biology

12.7K Aufrufe.  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.

Serie: 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.

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

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.

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 ...

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 ...

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

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 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.

Isolierung von hochwertigen atrialen und ventrikulären Myozyten der Maus für die simultane Messung von Ca2+ Transienten und L-Typ Calciumstrom

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.

Herstellung von Ratten-Skelettmuskelhomogenaten für Nitrat- und Nitritmessungen

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