Cette étude a été soutenue par National Heart, Lung, and Blood Institute Grant HL-103542, et le Prix Trailblazer émérite de la Fondation de recherche en anesthésie BCH à la PAC.

Préparation <ol><li> Préparez 1 M K-HEPES Stock Solution (ajuster le pH à 7,2 avec KOH). </li><li> Préparer M K-EGTA Stock Solution (ajuster le pH à 8,0 avec KOH) 0,5. </li><li> Préparez 1 M KH 2 PO 4 Stock Solution. </li><li> Préparez 1 M MgCl2 Solution mère. </li><li> Préparation du tampon d&#39;homogénéisation (pH 7,2), 300 mM de saccharose, 10 mM de HEPES-K, et 1 mM d&#39;EGTA-K. Conserver à 4 ° C. </li><li> Préparer le tampon de respiration 250 mM de saccharose, 2 mM de KH 2 PO 4, 10 mM MgCl 2, mM de tampon HEPES-K 20 (pH 7,2) et 0,5 mM de K-EGTA (pH 8,0). Conserver à 4 ° C. </li><li> Préparer la solution mère 10x PBS en dissolvant 80 g de NaCl, 2 g de KCl, 14,4 g de Na 2 HPO 4, et 2,4 g de KH 2 PO 4 dans 1 litre à double H 2 O distillée (pH 7,4). </li><li> Préparer 1x PBS à la pipette 100 ml 10x PBS dans 1 L à double H 2 O. distillée </li><li> Préparer subtilisine A Stock en pesant 4 mg de subtilisine A dansun tube à centrifuger de 1,5 ml. Conserver à -20 ° C jusqu&#39;à utilisation. </li><li> Préparer BSA Stock en pesant 20 mg de BSA dans un tube de 1,5 ml. Conserver à -20 ° C jusqu&#39;à utilisation. </li></ol> Isolement mitochondriale (figure 1) <ol><li> Immédiatement avant l&#39;isolement, la subtilisine A se dissoudre dans 1 ml de tampon d&#39;homogénéisation. </li><li> Immédiatement avant l&#39;isolement, la dissolution de BSA dans 1 ml de tampon d&#39;homogénéisation. </li><li> Recueillir deux échantillons de tissus frais en utilisant un poinçon 6 mm de l&#39;échantillon de biopsie et magasin en 1x PBS dans un tube à centrifuger conique de 50 ml sur la glace. </li><li> Transférez les deux poinçons 6 mm de tissu dans un tube de dissociation de C contenant 5 ml de glace froide homogénéisation tampon. </li><li> Homogénéiser le tissu en plaçant le tube de dissociation de C sur la dissociateur de tissus et sélectionnez le cycle de l&#39;isolement des mitochondries pré-établi (60 sec homogénéisation). </li><li> Retirer le tube de dissociation de C à un seau à glace. </li><li> Ajouter 250 pi de subtilisine A Solution mère à l&#39;l&#39;homogénat, mélanger par inversion et incuber l&#39;homogénat sur de la glace pendant 10 min. </li><li> Placer un filtre à mailles de 40 um sur un tube à centrifuger conique de 50 ml sur de la glace humide et pré-filtre avec le tampon d&#39;homogénéisation et de filtrage de l&#39;homogénat dans le tube à centrifuger conique de 50 ml sur de la glace. </li><li> Ajouter 250 ul de fraîchement préparé BSA Solution mère au filtrat et mélanger par inversion. Remarque: Ignorez cette étape si la détermination de la protéine mitochondriale est nécessaire. </li><li> Placer un filtre de 40 um sur un tube à centrifuger conique de 50 ml sur de la glace humide et pré-filtre avec le tampon d&#39;homogénéisation et de filtrage de l&#39;homogénat dans le tube à centrifuger conique de 50 ml sur de la glace. </li><li> Placer un filtre de 10 um sur un tube à centrifuger conique de 50 ml sur de la glace humide et pré-filtre avec le tampon d&#39;homogénéisation et de filtrage de l&#39;homogénat dans le tube à centrifuger conique de 50 ml sur de la glace. </li><li> Transférer le filtrat à deux pré-réfrigéré 1,5 ml microtubes à centrifuger et centrifuger à 9000 g pendant 10 min à4 ° C. </li><li> Retirer le surnageant et remettre en suspension et de combiner des pastilles dans 1 ml de tampon glacé respiration. </li></ol> ATP Assay Remarque: Pour déterminer l&#39;activité métabolique des mitochondries isolées d&#39;un dosage par luminescence de l&#39;ATP peut être effectuée en utilisant un kit de dosage de l&#39;ATP. Le protocole, réactifs et des étalons ont été fournis dans le kit de dosage. Un résumé de la procédure est décrite ci-dessous. <ol><li> Equilibrer les réactifs à la température ambiante. </li><li> Préparer 10 mM ATP Stock Solution en dissolvant culot ATP lyophilisée en 1170 ul d&#39;eau distillée deux fois. Rangez ATP Solution Stock standard et échantillons mitochondriaux préparés sur la glace. </li><li> Ajouter 5 ml de solution tampon de substrat à un flacon de solution de substrat lyophilisé. Mélanger délicatement et placer dans l&#39;obscurité. </li><li> Ajouter 100 ul de tampon de respiration à tous les puits de, un fond noir opaque, plaque de 96 puits. </li><li> Ajouter 10 ul de mitochondries des échantillons préparés dans chaque puits ofa noir, fond opaque, plaque de 96 puits. Note: Les échantillons sont étalés en triple. Inclure une ligne de normes et de trois puits pour le contrôle négatif (Respiration Buffer). </li><li> Ajouter 50 ul de solution de mammifère de la lyse des cellules dans chaque puits, y compris des normes et des contrôles. </li><li> Incuber la plaque à 37 ° C pendant 5 min sur un agitateur orbital à 125 tours par minute. </li><li> Au cours de l&#39;incubation préparer normes ATP dans des concentrations de 0,1 mM, 0,05 mM, 0,01 mM, 0,005 mM, 0,001 mM, et 0,0001 mM d&#39;ATP à partir de la solution mère mM ATP 10. normes de magasins sur la glace. </li><li> Après l&#39;incubation, ajouter 10 ul de standards de l&#39;ATP à des puits correspondant, comme indiqué sur la carte en forme de plaque. Note: Les normes sont effectuées en double. </li><li> Ajouter 50 ul de la solution de substrat reconstitué dans chaque puits. </li><li> Incuber la plaque à 37 ° C sur l&#39;agitation pendant 5 min à 125 rpm. </li><li> Ouvrez le logiciel Gen5 1.11 sur un ordinateur relié au spectrophotomètre. </li><li> Sous &quot;Créez un nouvel élément &quot;, cliquez sur&quot; Expérience &quot;. </li><li> Cliquez sur &quot;protocole par défaut&quot;. Cliquez sur &quot;Ok&quot;. </li><li> Dans la colonne de gauche, sélectionnez &quot;Protocole&quot;, puis &quot;procédure&quot;. </li><li> Sélectionnez &quot;Delay&quot;. Situé à 0:10:00. Cliquez sur &quot;Ok&quot;. </li><li> Sélectionnez &quot;Lire&quot;. Sélectionnez &quot;Luminescence&quot; de menu déroulant. Définissez les autres paramètres: Lire le type Endpoint, temps d&#39;intégration 0: 01.00 MM: SS.SS, Filtre Règle 1, émission Hole, Optique Position Haut, sensibilité 100, Top sonde Décalage vertical 1.00 mm. </li><li> Lorsque la plaque est prêt à être analysé, cliquez sur l&#39;icône &quot;Lire Plate&quot; sur la rangée du haut. Cliquez sur &quot;Lire&quot;. Lorsque le spectrophotomètre s&#39;ouvre, placez la plaque sur le plateau, le puits A1 dans le coin supérieur gauche. Une boîte avec la température va s&#39;ouvrir. Cliquez sur &quot;Lire&quot;. Remarque: Des valeurs plus élevées sont en corrélation avec les niveaux d&#39;ATP accrus et l&#39;activité métabolique plus élevé. </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. 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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. 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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. 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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>

Tous les animaux ont été traités conformément aux directives institutionnelles actuelles. Nous n&#39;avons pas d&#39;intérêts financiers concurrents de divulguer.

Pour isoler avec succès mitochondries qui utilisent ce protocole, il est essentiel de garder toutes les solutions et les échantillons de tissus sur la glace pour conserver la viabilité mitochondriale. Même lorsqu&#39;il est maintenu sur la glace, les mitochondries isolées présenteront une diminution de l&#39;activité fonctionnelle au fil du temps 19. Nous recommandons que toutes les solutions et les ajouts être pré-préparés. Nous avons pré-peser et stocker subtilisine A à 4 mg aliquotes de 1,5 ml...

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

Les mitochondries existent dans toutes les cellules du corps à l&#39;exception des globules rouges et sont impliqués dans un grand nombre de processus cellulaires et métaboliques importantes 1-4. En raison de ces nombreuses fonctions, dommage mitochondrial peut avoir des effets néfastes 3. Afin d&#39;étudier la fonction mitochondriale et la dysfonction plusieurs méthodes d&#39;isolement des mitochondries ont été décrits. Les premiers comptes publiés de mitochondrial date de l&#39;isolement à la 1940 5-8. Le premier essai a démontré documenté isolement des mitochondries par broyage du tissu hépatique dans un mortier puis par centrifugation dans une solution de sel à faible vitesse 5,8. Plus tard, d&#39;autres groupes étendus sur la procédure d&#39;origine et ont démontré fractionnement du tissu sur la base de la centrifugation différentielle 8.6. Ces premières méthodes ont constitué la base des techniques actuelles qui intègrent souvent homogénéisation et / ou centrifugation différentielle 9-15. Le nombre de homogenizatiet les étapes de centrifugation varie selon les protocoles. Ces étapes répétitives d&#39;augmenter le temps pour l&#39;isolement des mitochondries et finalement réduisent la viabilité. En outre, l&#39;homogénéisation manuel peut provoquer des dommages et incohérentes mitochondriales résultats s&#39;il n&#39;est pas correctement contrôlée 10,16. Récemment, nous avons utilisé l&#39;homogénéisation et centrifugation différentielle pour isoler les mitochondries pour la transplantation dans le tissu myocardique 17,18. Cette procédure d&#39;isolement longue besoin d&#39;environ 90 minutes et l&#39;applicabilité clinique de cette méthode est donc limitée. Pour permettre une utilisation thérapeutique dans le traitement aigu et clinique chirurgicale nous avons mis au point un procédé d&#39;isolement des mitochondries rapide qui peut être effectué en moins de 30 min. Les principaux avantages de ce protocole sont que la dissociation des tissus uniformisée permet d&#39;uniforme et cohérente homogénéisation du tissu qui n&#39;est pas facile à réaliser avec homogénéisation manuel. Dans suppion, l&#39;utilisation de la filtration différentielle à la place de la centrifugation différentielle élimine beaucoup de temps et répétition des étapes de centrifugation permettant l&#39;isolement des mitochondries plus rapide compétentes hautement purifiés, viables et de respiration. La capacité à isoler les mitochondries compétentes viables et de respiration en moins de 30 minutes permet d&#39;applicabilité clinique. Ce protocole d&#39;isolement a un potentiel pour une utilisation en chirurgie coronaire pontage aorto-greffe (CABG), et d&#39;autres procédures thérapeutiques.

<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

Méthodes d&#39;isolement des mitochondries décrites précédemment par centrifugation différentielle et / ou gradient de Ficoll centrifugation nécessitent 60 à 100 min pour terminer. Nous décrivons un procédé pour l&#39;isolement rapide de mitochondries à partir de biopsies de mammifère en utilisant un dissociateur tissulaire commercial et filtration différentielle. Dans ce protocole, l&#39;homogénéisation manuel est remplacé par un cycle d&#39;homogénéisation normalisée du dissociateur tissulaire. Cela permet d&#39;homogénéisation uniforme et cohérente de tissu qui n&#39;est pas facile à réaliser avec homogénéisation manuelle. Après dissociation des tissus, le broyat est filtré à travers des filtres de maille de nylon, ce qui élimine les étapes de centrifugation répétitives. En conséquence, l&#39;isolement des mitochondries peut être effectuée en moins de 30 min. Ce protocole d&#39;isolement rendement d&#39;environ 2 x 10 10 mitochondries compétentes viables et la respiration de 0,18 ± 0,04 g (poids humide) de l&#39;échantillon de tissu.

A la figure décrivant les étapes de la procédure dans l&#39;isolement des mitochondries des tissus à l&#39;aide de dissociation et de filtration différentielle est représentée sur la Figure 1. Temps de procédure total est inférieur à 30 min. Des échantillons de tissus ont été obtenus en utilisant un poinçon de biopsie de 6 mm. poids des tissus a été de 0,18 ± 0,04 g (poids humide). Le nombre des mitochondries isolées tel que déterminé par la taille des pa...

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

Procédé pour l&#39;isolement rapide de mitochondries à partir de biopsies de tissu de mammifère est décrite. foie de rat ou des préparations musculaires squelettiques ont été homogénéisés avec un dissociateur tissu commercial et les mitochondries ont été isolées par filtration différentielle à travers des filtres en filet de nylon. Temps d&#39;isolement des mitochondries est &lt;30 min par rapport à 60 - 100 min en utilisant des méthodes alternatives.

Isolement rapide et la purification des mitochondries pour la transplantation par la dissociation des tissus et différencié 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 vues.  Beth Israel Deaconess Medical Center and Harvard Medical School.  Procédé pour l'isolement rapide de mitochondries à partir de biopsies de tissu de mammifère est décrite. foie de rat ou des préparations musculaires squelettiques ont été homogénéisés avec un dissociateur tissu commercial et les mitochondries ont été isolées par filtration différentielle à travers des filtres en filet de nylon. Temps d'isolement des mitochondries est 

Vidéo: 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

L&#39;isolement et la transplantation de cellules souches hématopoïétiques (CSH)

Recherche

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.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

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.

Purification des mitochondries de cellules de levure

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.

Isolement et purification de Drosophile Neurones périphériques en triant bille magnétique

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.

Isolement de la chromatine par purification d&#39;ARN (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.

Immobilisation planaire, l&#39;irradiation partielle, et la transplantation de tissus

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;

L&#39;isolement, la culture, et la transplantation de cellules satellites du muscle

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.

Isolement et analyse fonctionnelle des mitochondries de cellules cultivées et de tissus de souris

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 Isolement des acides nucléiques: Une méthode d&#39;extraction de l&#39;ADN simple et rapide

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.