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>

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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

37.9K 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

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolierung und Reinigung von Kinesin aus Drosophila Embryonen

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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 ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protokoll für die Isolierung von primären humanen Hepatozyten und entsprechende Hauptpopulationen von Nicht-parenchymale Leberzellen

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...