Diese Studie wurde von National Heart, Lung, and Blood Institute Zuschuss HL-103542 und der BCH Anästhesie Research Foundation Distinguished Trailblazer Award an GAP unterstützt.

Vorbereitung <ol><li> Bereiten 1 M K-HEPES-Stammlösung (einstellen mit KOH pH-Wert auf 7.2). </li><li> Bereiten 0,5 M K-EGTA-Stammlösung (einstellen mit KOH pH-Wert auf 8,0). </li><li> Bereiten 1 M KH 2 PO 4 Stock Lösung. </li><li> Bereiten 1 M MgCl 2-Stammlösung. </li><li> Vorbereitung Homogenisierungspuffer (pH 7,2) 300 mM Saccharose, 10 mM K-HEPES, 1 mM K-EGTA. Lagerung bei 4 ° C. </li><li> Vorbereitung Respiration Puffer 250 mM Saccharose, 2 mM KH 2 PO 4, 10 mM MgCl 2, 20 mM K-HEPES-Puffer (pH 7,2) und 0,5 mM K-EGTA (pH 8,0). Lagerung bei 4 ° C. </li><li> Vorbereitung 10x PBS-Stammlösung hergestellt durch Lösen von 80 g NaCl, 2 g KCl, 14,4 g Na 2 HPO 4 und 2,4 g KH 2 PO 4 in 1 l doppelt destilliertem H 2 O (pH 7,4). </li><li> Bereiten 1x PBS pipettiert 100 ml 10x PBS in 1 l doppelt destilliertem H 2 O. </li><li> Bereiten Subtilisin A Stock durch Auswiegen 4 mg Subtilisin A inin ein 1,5 ml Mikrozentrifugenröhrchen. Lagerung bei -20 ° C bis zur Verwendung. </li><li> Vorbereitung BSA Vektor durch Einwaage von 20 mg BSA in ein 1,5 ml Mikrozentrifugenröhrchen. Lagerung bei -20 ° C bis zur Verwendung. </li></ol> Mitochondriale Isolation (Abbildung 1) <ol><li> Unmittelbar vor der Isolation, auflösen Subtilisin A in 1 ml Homogenisierungspuffer. </li><li> Unmittelbar vor der Isolation, auflösen BSA in 1 ml Homogenisierungspuffer. </li><li> Sammeln Sie zwei frische Gewebeproben mit einem 6 mm Biopsieprobe Punsch und speichern in 1x PBS in einem 50 ml konischen Zentrifugenröhrchen auf Eis. </li><li> Übertragen Sie die zwei 6 mm Stempeln von Gewebe zu einer Dissoziation C-Rohr mit 5 ml eiskaltem Homogenisierungspuffer. </li><li> Homogenisieren des Gewebes durch Anpassen der Dissoziation C-Rohr auf das Gewebe Dissociator und wählen Sie den voreingestellten mitochondrialen Isolierung Zyklus (60 Sekunden Homogenisierung). </li><li> Entfernen Sie die Dissoziation C-Rohr zu einem Eimer Eis. </li><li> Fügen Sie 250 ul Subtilisin A Stock Lösung, um dieHomogenat, mischen durch Umdrehen und Inkubation des Homogenats auf Eis für 10 min. </li><li> Legen Sie ein 40 um-Sieb-Filter auf eine 50 ml konischen Zentrifugenröhrchen auf Eis und nassen vorge dem Filter mit Homogenisieren Buffer und filtern das Homogenat in die 50 ml konischen Zentrifugenröhrchen auf Eis. </li><li> Fügen Sie 250 ul frisch zubereitet BSA-Stammlösung zu dem Filtrat und mischen durch Umdrehen. Hinweis: Lassen Sie diesen Schritt, wenn mitochondrialen Proteinbestimmung erforderlich ist. </li><li> Legen Sie eine 40-um-Filter auf eine 50 ml konischen Zentrifugenröhrchen auf Eis und nassen vorge dem Filter mit Homogenisieren Buffer und filtern das Homogenat in die 50 ml konischen Zentrifugenröhrchen auf Eis. </li><li> Legen Sie eine 10-um-Filter auf eine 50 ml konischen Zentrifugenröhrchen auf Eis und nassen vorge dem Filter mit Homogenisieren Buffer und filtern das Homogenat in die 50 ml konischen Zentrifugenröhrchen auf Eis. </li><li> Das Filtrat zwei vorgekühlte 1,5 ml Mikrozentrifugenröhrchen und Zentrifuge bei 9000 × g für 10 min bei4 ° C. </li><li> Überstand entfernen und wieder aussetzen und kombinieren Pellets in 1 ml eiskaltem Puffer Atmung. </li></ol> ATP-Assay Anmerkung: Zur Bestimmung der metabolischen Aktivität von isolierten Mitochondrien eine ATP-Lumineszenz-Assay kann unter Verwendung eines ATP-Assay-Kits durchgeführt werden. Das Protokoll, wurden Reagenzien und Standards in der Assay-Kit geliefert. Eine Zusammenfassung des Verfahrens wird nachfolgend beschrieben. <ol><li> Ins Gleichgewicht Kit-Reagenzien auf RT. </li><li> Herstellung von 10 mM ATP-Stammlösung durch Auflösen von gefriergetrockneten ATP Pellet in 1.170 ul doppelt destilliertem Wasser. Speichern ATP-Standardstammlösung und bereit mitochondrialen Proben auf Eis. </li><li> 5 ml Substratpuffer Lösung für ein Fläschchen lyophilisierte Substrat-Lösung. Vorsichtig mischen und legen in der Dunkelheit. </li><li> 100 l Puffer Atmung zu allen Vertiefungen einer schwarzen, undurchsichtigen Boden, 96 Well-Platte. </li><li> Fügen Sie 10 ul der Mitochondrien aus den vorbereiteten Proben in jede Vertiefung ofa schwarz, undurchsichtig unten, 96-Well-Platte. Hinweis: Die Proben werden in dreifacher Ausfertigung überzogen. Fügen Sie eine Zeile für Standards und drei Brunnen für die Negativkontrolle (Atmung Buffer). </li><li> Fügen Sie 50 ul von Säugetierzell Lyse-Lösung in alle Vertiefungen, einschließlich der Normen und Kontrollen. </li><li> Die Platte bei 37 ° C für 5 min auf einem Orbitalschüttler bei 125 Umdrehungen pro Minute. </li><li> Während der Inkubation herzustellen ATP-Standards in Konzentrationen von 0,1 mM, 0,05 mM, 0,01 mM, 0,005 mM, 0,001 mM und 0,0001 mM ATP aus 10 mM ATP-Stammlösung. Shop-Standards auf Eis. </li><li> Nach der Inkubation werden 10 ul der ATP-Standards mit entsprechenden Vertiefungen wie auf der Platte Karte angezeigt. Anmerkung: Standards werden in doppelter Ausführung durchgeführt. </li><li> Dann werden 50 ul des rekonstituierten Substratlösung in jede Vertiefung. </li><li> Inkubieren der Platte bei 37 ° C auf dem Schüttler für 5 Minuten bei 125 Upm. </li><li> Offene Gen5 1.11-Software auf einem Computer mit dem Spektrophotometer verbunden ist. </li><li> Unter &quot;Neues Element erstellen &quot;, klicken Sie auf&quot; Experiment &quot;. </li><li> Klicken Sie auf &quot;Standardprotokoll&quot;. Klicken Sie auf &quot;OK&quot;. </li><li> In der linken Spalte, wählen Sie &quot;Protokoll&quot;, dann &quot;Vorgehensweise&quot;. </li><li> Wählen Sie &quot;Delay&quot;. Eingestellt, 00.10.00. Klicken Sie auf &quot;OK&quot;. </li><li> Wählen Sie &quot;Lesen&quot;. Wählen Sie &quot;Lumineszenz&quot; aus der Drop-Down-Menü. Passen Sie andere Einstellungen auf die folgenden: Lesen Typ Endpoint, Integrationszeit 0: 01.00 MM: SS.ss, Filter-Sets 1, Emission Loch, Optik Top Position, Empfindlichkeit 100, Offset Top Probe Vertikal 1,00 mm. </li><li> Wenn die Platte ist bereit, analysiert werden, klicken Sie auf das Symbol &quot;Lesen Plate&quot; in der oberen Reihe. Klicken Sie auf &quot;Lesen&quot;. Wenn das Spektralphotometer öffnet, legen Sie die Platte in das Fach mit gut A1 in der oberen linken Ecke. Eine Box mit der Temperatur wird geöffnet. Klicken Sie auf &quot;Lesen&quot;. Hinweis: Höhere Werte korrelieren mit erhöhten ATP-Spiegel und höhere Stoffwechselaktivität. </li></ol>

<ol>
	<li>van Loo, G., Saelens, X., van Gurp, M., MacFarlane, M., Martin, S. J., Vandenabeele, P. <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+mitochondrial+factors+in+apoptosis:+a+Russian+roulette+with+more+than+one+bullet.">The role of mitochondrial factors in apoptosis: a Russian roulette with more than one bullet.</a> Cell Death Differ. 9 (10), 1031-1042 (2002).</li><li>Szabadkai, G., 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=Mitochondria:+the+hub+of+cellular+Ca2++signaling.">Mitochondria: the hub of cellular Ca2+ signaling.</a> Physiology (Bethesda). 23, 84-94 (2008).</li><li>Chan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+Dynamic+Organelles+in+Disease,+Aging,+and+Development.">Mitochondria: Dynamic Organelles in Disease, Aging, and Development.</a> Cell. 125 (7), 1241-1252 (2006).</li><li>Picard, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+structure+and+function+are+disrupted+by+standard+Isolation+methods.">Mitochondrial structure and function are disrupted by standard Isolation methods.</a> PLoS ONE. 6 (3), e18317 (2011).</li><li>Bensley, R. R., Hoerr, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+cell+structure+by+freeze-drying+method;+preparation+and+properties+of+mitochondria.">Studies on cell structure by freeze-drying method; preparation and properties of mitochondria.</a> Anat Rec. 60, 449-455 (1934).</li><li>Claude, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fractionation+of+mammalian+liver+cells+by+differential+centrifugation+II.+Experimental+procedures+and+results.">Fractionation of mammalian liver cells by differential centrifugation II. Experimental procedures and results.</a> J Exp Med. 84 (1), 61-89 (1946).</li><li>Hogeboom, H., Schneider, W. C., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+studies+of+mammalian+tissues;+isolation+of+intact+mitochondria+from+rat+liver;+some+biochemical+properties+of+mitochondria+and+submicroscopic+particulate+material.">Cytochemical studies of mammalian tissues; isolation of intact mitochondria from rat liver; some biochemical properties of mitochondria and submicroscopic particulate material.</a> J Biol Chem. 172 (2), 619-635 (1948).</li><li>Ernster, L., Schtaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria:+a+historical+Review.">Mitochondria: a historical Review.</a> J Cell Biol. 91 (3 Pt 2), 227s-255s (1981).</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>Schmitt, 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=A+semi-automated+method+for+isolating+functionally+intact+mitochondria+from+cultured+cells+and+tissue+biopsies.">A semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies.</a> Anal Biochem. 443 (1), 66-74 (2013).</li><li>Fern&#225;ndez-Vizarra, E., Ferr&#237;n, G., P&#233;rez-Martos, A., Fern&#225;ndez-Silva, P., Zeviani, M., Enr&#237;quez, 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+mitochondria+for+biogenetical+studies:+An+update.">Isolation of mitochondria for biogenetical studies: An update.</a> Mitochondrion. 10 (3), 253-262 (2010).</li><li>Graham, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3,+Unit+3.3,+Isolation+of+mitochondria+from+tissues+and+cells+by+differential+centrifugation.">Chapter 3, Unit 3.3, Isolation of mitochondria from tissues and cells by differential centrifugation.</a> Curr Protoc Cell Biol. , (2001).</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 (2), 287-295 (2007).</li><li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells.">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat Protoc. 4 (11), 1582-1590 (2009).</li><li>Gostimskaya, I., Galkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+highly+coupled+rat+heart+mitochondria.">Preparation of highly coupled rat heart mitochondria.</a> J Vis Exp. (43), (2010).</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 (2), 213-223 (2011).</li><li>Masuzawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury.">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> American J Physiol Heart and Circ Physiol. 304 (7), (2013).</li><li>McCully, J. 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=Injection+of+isolated+mitochondria+during+early+reperfusion+for+cardioprotection.">Injection of isolated mitochondria during early reperfusion for cardioprotection.</a> American J Physiol Heart and Circ Physiol. 296 (1), 94-105 (2009).</li><li>Olson, M. S., Von Korff, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+endogenous+substrates+of+isolated+rabbit+heart+mitochondria+during+storage.">Changes in endogenous substrates of isolated rabbit heart mitochondria during storage.</a> J Biol Chem. 242 (2), 325-332 (1967).</li><li>Diepart, C., 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=Comparison+of+methods+for+measuring+oxygen+consumption+in+tumor+cells+in+vitro.">Comparison of methods for measuring oxygen consumption in tumor cells in vitro.</a> Anal Biochem. 396 (2), 250-256 (2010).</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>

Alle Tiere wurden in Übereinstimmung mit den geltenden Richtlinien des Instituts behandelt. Wir haben keine finanziellen Interessen im Wettbewerb offen zu legen.

Um erfolgreich zu isolieren Mitochondrien mit diesem Protokoll ist es wichtig, alle Lösungen und Gewebeproben auf Eis zu halten, um die mitochondriale Lebensfähigkeit bewahren. Auch wenn auf Eis gehalten wird isolierten Mitochondrien eine Abnahme der funktionellen Aktivität im Laufe der Zeit 19 aufweisen. Wir empfehlen, dass alle Lösungen und Ergänzungen vor, vorbereitet werden. Wir voraus wiegen und zu speichern Subtilisin A in 4 mg Aliquots in 1,5 ml Mikrozentrifugenröhrchen und speichern sie bei -20 ...

A correction was made to: <a href="http://www.jove.com/video/51682">Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.</a> An optional step was added to step 7 of the &#39;Mitochondrial Isolation&#39; section of the protocol. The step is:
1. Optional in the case that the tissue is fibrous: Centrifuge the solution at 750 x g for 4 min.

A correction was made to: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration.

Erratum: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Mitochondrien existieren in jeder Zelle des Körpers mit Ausnahme der roten Blutzellen und in einer Vielzahl von wichtigen zellulären Stoffwechselprozesse und 1-4 involviert. Aufgrund dieser vielen Funktionen können mitochondriale Schäden schädlichen Wirkungen 3 aufweisen. Um die Funktion der Mitochondrien und mitochondriale Dysfunktion mehrere Isolationsmethoden zu untersuchen, wurden beschrieben. Die frühesten veröffentlichten Konten von mitochondrialen Isolierung Datum auf den 1940er 5-8. Der erste dokumentierte Versuch demonstriert mitochondrialen Isolierung durch Schleifen Lebergewebe in einem Mörser, gefolgt von Zentrifugation in einer Salzlösung, bei niedriger Geschwindigkeit 5,8. Später erweiterte anderen Gruppen auf dem ursprünglichen Verfahren und demonstrierte Gewebe Fraktionierung basierend auf differentielle Zentrifugation 6-8. Diese frühen Methoden bildeten die Grundlage der aktuellen Techniken, die oft zu integrieren Homogenisierung und / oder differenzielle Zentrifugation 9-15. Die Anzahl der homogenizatiauf und Zentrifugationsschritte variiert zwischen den Protokollen. Diese sich wiederholenden Schritten erhöhen die Zeit für mitochondriale Isolation und letztlich reduzieren Lebensfähigkeit. Darüber hinaus können manuelle Homogenisierung mitochondriale Schäden und inkonsistenten Ergebnissen führen, wenn nicht richtig gesteuert 10,16. Kürzlich haben wir Homogenisierung und differentielle Zentrifugation Mitochondrien für die Transplantation in Herzmuskelgewebe 17,18 isolieren. Dieser langwierige Isolierungsverfahren benötigt ungefähr 90 min, und die klinische Anwendbarkeit dieses Verfahrens war daher begrenzt. Zur therapeutischen Verwendung bei akuten klinischen und chirurgischen Behandlung zu ermöglichen haben wir eine schnelle mitochondrialen Isolierungsverfahren, die in weniger als 30 min durchgeführt werden kann, entwickelt. Die wichtigsten Vorteile dieses Protokolls sind, dass standardisierte Gewebedissoziation ermöglicht einheitliche und konsequente Homogenisierung von Gewebe, das wird nicht leicht mit Hand Homogenisierung erreicht. In additIonen, die Verwendung von Differentialfiltration anstelle der differentielle Zentrifugation eliminiert zeitraubende und wiederholte Zentrifugation Schritte ermöglichen eine schnellere Isolierung von hoch gereinigten, lebensfähige und kompetente Atmung Mitochondrien. Die Fähigkeit, tragfähige und Atmung zuständige Mitochondrien in weniger als 30 min zu isolieren können für die klinische Anwendbarkeit. Diese Isolation Protokoll hat Potenzial für den Einsatz in koronarer Bypass-Chirurgie (CABG) und andere Therapieverfahren.

<table border="0" cellpadding="0" cellspacing="0" width="933">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:377px;">Sucrose</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:147px;">84100</td>
			<td style="width:229px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Substilsin A</td>
			<td>Sigma Aldrich</td>
			<td>P5380</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td>Sigma Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Sigma Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S6191</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>P2173</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:377px;">ATPlite Luminescence Assay System, 
			1000 Assay Kit</td>
			<td>Perkin Elmer</td>
			<td>6016941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mL Conical Tubes</td>
			<td>BD</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 &#956;m Nylon Filters</td>
			<td>BD&#160;</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GentleMACS C tube</td>
			<td>Miltenyl Biotech</td>
			<td>120-005-331</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Eppendorf tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 mm biopsy punch</td>
			<td>Miltex</td>
			<td>33-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 &#956;m Pluristrainer</td>
			<td>Pluriselect</td>
			<td>43-500-10-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Centrifuge 5415C</td>
			<td>Marshall Scientific</td>
			<td>EP-5415C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GentleMACS Dissociator&#160;</td>
			<td>Miltenyl Biotech</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96-well plates, tissue culture treated</td>
			<td>VWR</td>
			<td>82050-732</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rotomax 120 orbital shaker</td>
			<td>Heidolph</td>
			<td>544-41200-00</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Synergy H4 Hybrid Multi-Mode 
			&#160;Microplate Reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oxytherm System</td>
			<td>Hansatech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration

Zuvor beschriebenen mitochondrialen Isolationsverfahren unter Verwendung differentieller Zentrifugation und / oder Ficoll-Gradienten-Zentrifugation erfordert 60 bis 100 min in Anspruch. Wir beschreiben ein Verfahren zur schnellen Isolierung von Mitochondrien aus Säugetierbiopsien unter Verwendung eines kommerziellen Gewebe Dissociator und Differentialfiltration. In diesem Protokoll ist eine manuelle Homogenisierung mit standardisierten Zyklus der Homogenisierung des Gewebes Dissociator ersetzt. Dies ermöglicht eine einheitliche und konsequente Homogenisierung von Gewebe, das wird nicht leicht mit Hand Homogenisierung erreicht. Nach Gewebedissoziation wird das Homogenat durch Nylon-Mesh-Filter, die sich wiederholenden Zentrifugationsschritten beseitigen gefiltert. Als Ergebnis kann die mitochondriale Isolierung in weniger als 30 min durchgeführt werden. Diese Isolation Protokoll ergibt etwa 2 x 10 10 lebensfähige und Atmung zuständige Mitochondrien von 0,18 ± 0,04 g (Nassgewicht) Gewebeprobe.

Eine Figur skizziert die Verfahrensschritte bei der Isolierung von Mitochondrien mit Gewebedissoziation und Differenz Filtration ist in Abbildung 1 dargestellt. Gesamtverfahrensdauer weniger als 30 min. Gewebeproben wurden mit einem 6 mm-Biopsie Punch erhalten. Gewebegewicht betrug 0,18 ± 0,04 g (Feuchtgewicht). Die Anzahl der Mitochondrien isoliert, wie durch die Teilchengröße Zählung bestimmt war 2,4 x 10 10 ± 0,1 x 10 10 Mitochondrien für Skele...

Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Ein Verfahren zur schnellen Isolierung von Mitochondrien aus Säugergewebebiopsien beschrieben. Rattenleber-oder Skelettmuskelpräparate wurden mit einem kommerziellen Gewebe Dissociator homogenisiert und Mitochondrien wurden durch Differentialfiltration durch Nylon-Mesh-Filter isoliert. Mitochondriale Isolation Zeit &lt;30 min im Vergleich zu 60 bis 100 min mit alternativen Methoden.

Schnelle Isolierung und Reinigung von Mitochondrien für die Transplantation von Gewebe-Dissoziation und Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

rapid-isolation-purification-mitochondria-for-transplantation-tissue

Research

JoVE Journal

Biology

37.7K Aufrufe.  Beth Israel Deaconess Medical Center and Harvard Medical School.  Ein Verfahren zur schnellen Isolierung von Mitochondrien aus Säugergewebebiopsien beschrieben. Rattenleber-oder Skelettmuskelpräparate wurden mit einem kommerziellen Gewebe Dissociator homogenisiert und Mitochondrien wurden durch Differentialfiltration durch Nylon-Mesh-Filter isoliert. Mitochondriale Isolation Zeit 

Serie: Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolierung und Transplantation von hämatopoetischen Stammzellen (HSZ)

Forschung

Biologie

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Reinigung von Mitochondrien aus Hefezellen

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolierung und Reinigung von Drosophila Peripheren Neuronen durch Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Chromatin Isolierung von RNA-Reinigung (Chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Planarien Immobilisierung, partielle Bestrahlung, und Gewebetransplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolierung, Kultur und Transplantation von Muskel-Satelliten-Zellen

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Isolierung und funktionale Analyse von Mitochondrien aus kultivierten Zellen und Maus-Gewebe

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Filtration Isolierung von Nukleinsäuren: Eine einfache und schnelle DNA-Extraktionsmethode

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

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.