Name: rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration
Uploaded: 2014-09-06T14:00:00
Description: Watch this Scientific Journal Video about Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration at JoVE.com

Denne undersøgelse blev støttet af National Heart, Lung, og Blood Institute Grant HL 103542, og The BCH Anæstesi Forskningsfonds Distinguished Trailblazer Award til den fælles landbrugspolitik.

Forberedelse <ol><li> Forbered 1 M K-HEPES stamopløsningen (justering af pH til 7,2 med KOH). </li><li> Forbered 0,5 M K-EGTA stamopløsningen (justering af pH til 8,0 med KOH). </li><li> Forbered 1 M KH 2 PO 4 stamopløsning. </li><li> Forbered 1 M MgCl2 stamopløsning. </li><li> Forbered Homogenisering buffer (pH 7,2) 300 mM saccharose, 10 mM K-HEPES og 1 mM K-EGTA. Opbevares ved 4 ° C. </li><li> Forbered respiration buffer 250 mM saccharose, 2 mM KH 2PO 4, 10 mM Mg...

<ol>
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For med held at isolere mitokondrier ved hjælp af denne protokol er det vigtigt at holde alle løsninger og vævsprøver på is for at bevare mitokondrie levedygtighed. Selv når opretholdes på is, vil isolerede mitokondrier udviser et fald i funktionel aktivitet over tid 19. Vi anbefaler, at alle løsninger og tilføjelser være allerede forberedt. Vi pre-veje og gemme Subtilisin A i 4 mg portioner i 1,5 ml mikrocentrifugerør og opbevare dem ved -20 ° C. Tilsvarende BSA forvejet og opbevares i 20 mg port...

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

Findes mitokondrier i hver celle i kroppen undtagen røde blodlegemer og er involveret i en lang række vigtige cellulære og metaboliske processer 1-4. På grund af disse mange funktioner, kan skade på mitokondrier have skadelige virkninger 3. For at undersøge mitokondriefunktion og dysfunktion flere mitochondriale isoleringsmetoder er blevet beskrevet. De tidligste offentliggjorte regnskaber mitokondrie isolation dato til 1940&#39;erne 5-8. Den første dokumenterede forsøg påvist mitokondrie isolering ved formaling levervæv i en morter efterfulgt af centrifugering i en saltopløsning ved lav hastighed 5,8. Senere andre grupper uddybet den oprindelige procedure og demonstreret væv fraktionering baseret på differentiel centrifugering 6-8. Disse tidlige metoder dannede grundlag af de nuværende teknikker, som ofte inkorporerer homogenisering, og / eller differential centrifugering 9-15. Antallet af homogenizatipå og centrifugering trin varierer mellem protokoller. Disse gentagne trin øg tidsrummet for mitokondrie isolation og i sidste ende reducere levedygtighed. Hertil kommer, kan manuel homogenisering forårsage skade på mitokondrier og uensartede resultater, hvis ikke ordentligt kontrolleret 10,16. For nylig brugte vi homogenisering og differentialcentrifugering at isolere mitokondrier til transplantation i myokardievæv 17,18. Denne langvarige isolation, der kræves cirka 90 minutter, og den kliniske anvendelighed af denne metode var derfor begrænset. For at give mulighed for akut terapeutisk anvendelse i klinisk og kirurgisk behandling har vi udviklet en hurtig mitokondrie isolation procedure, der kan udføres i mindre end 30 min. De største fordele ved denne protokol er, at standardiserede væv dissociation tillader ensartet og konsekvent homogenisering af væv, som ikke er nemt at opnå med manuel homogenisering. I Addition, brug af differentieret filtrering i stedet for differentialcentrifugering eliminerer tidskrævende og gentagne centrifugeringstrin giver mulighed for hurtigere isolering af højt oprensede, levedygtige og respiration kompetente mitokondrier. Evnen til at isolere levedygtige og åndedræt kompetente mitokondrier i mindre end 30 min giver mulighed for klinisk anvendelighed. Denne isolation protokol har potentiale til brug i koronararterie bypass-kirurgi (CABG) og andre terapeutiske procedurer.

<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

Tidligere beskrevne mitokondrielle isoleringsmetoder anvender differentiel centrifugering og / eller Ficoll-gradientcentrifugering kræve 60 til 100 minutter for at fuldføre. Vi beskriver en metode til hurtig isolering af mitokondrier fra pattedyr biopsier ved hjælp af en kommerciel væv dissociator og differentieret filtrering. I denne protokol, er manuel homogenisering erstattet med vævet dissociator standardiserede homogenisering cyklus. Dette giver mulighed for en ensartet og konsekvent homogenisering af væv, der ikke let opnås med manuel homogenisering. Efter væv dissociation er homogenat filtreres gennem nylon mesh filter, som fjerner repetitive centrifugeringstrin. Som et resultat, kan mitokondrie isolering udføres i mindre end 30 min. Denne isolation protokol giver ca. 2 x 10 10 levedygtige og åndedræt kompetente mitokondrier fra 0,18 ± 0,04 g (våd vægt) vævsprøve.

En figur skitserer de proceduremæssige skridt i isoleringen af mitokondrier hjælp af væv dissociation og differentieret filtrering er vist i figur 1. Samlet processuelle tid er mindre end 30 min. Vævsprøver blev opnået ved anvendelse af en 6 mm biopsitang. Tissue vægt var 0,18 ± 0,04 g (våd vægt). Antallet af mitokondrier isoleret som bestemt ved partikelstørrelse optælling var 2,4 x 10 10 ± 0.1 x 10 10 mitokondrier for skeletmuskulatur og ...

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

En metode til hurtig isolering af mitokondrier fra pattedyr vævsbiopsier beskrives. Rotte lever eller skeletmuskulaturen præparater blev homogeniseret med en kommerciel væv dissociator og mitokondrier blev isoleret ved differential filtrering gennem nylon mesh filtre. Mitokondrie isolation tid er &lt;30 min sammenlignet med 60-100 minutter ved hjælp af alternative metoder.

Hurtig isolering og oprensning af mitokondrier til transplantation Ved Tissue Dissociation og differentieret Filtrering

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

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