Ce travail a été soutenu par une subvention de l&#39;Institut national des yeux (R01 EY12998) à FH Andrade.

1. Préparation des tampons <ol><li> Allumez 5804R centrifugeuse et fixé à 4 ° C. Tourner sur le bain d&#39;eau Isotemp 3006D et mettre à 37 ° C. </li><li> Préparer les solutions suivantes avant l&#39;isolement du muscle: <ul><li> PBS: dissoudre tampon phosphate salin (PBS) comprimés dans de l&#39;eau distillée (5 comprimés / litre). Mélangez bien. </li><li> PBS + 10 mM EDTA: Pour préparer une solution de 100 ml, ajouter 2 ml d&#39;EDTA 500 mM à 98 ml de PBS. </li><li> 8X tampon Mitochondries: 10,28 g de saccharose pour une concentration finale de 0,6 M, 400 mg d&#39;acides gras libres de sérum albumine bovine (BSA) pour une concentration finale de 0,8%, 2,08 g d&#39;HEPES pour une concentration finale de 160 mM, pH à 7,4 et QS à 50 ml d&#39;eau distillée. </li><li> Une mémoire tampon d&#39;isolement (IB1): Ajouter 200 ul d&#39;EDTA 500 mM pour une concentration finale de 10 mM, 0,392 g de D-mannitol pour une concentration finale de 215 mM, 1,25 ml de 8X, pH à 7,4 mitochondries et QS à 10 ml d&#39;eau distillée. </li><li> 2 tampons Isolement (IB2): Ajouter 60 ul de 500 mM EGTA pour une concentration finale de 3 mM, 0,392 g de D-mannitol pour une concentration finale de 215 mM, 1,25 ml de 8X, pH à 7,4 mitochondries et QS à 10 ml d&#39;eau distillée. </li><li> Tampon expérimental (EB): Ajouter 100 ul de 500 mM de MgCl 2 pour une concentration finale de 5 mM, 0,392 g de D-mannitol pour une concentration finale de 215 mm, 25 pl de 2,5 mM KH 2 PO 4 pour une concentration finale de 6,25 uM, 2 pl de 100 mM EGTA pour une concentration finale de 20 uM, 1,25 ml de tampon mitochondrial, le pH à 7,4 et QS à 10 ml d&#39;eau distillée. </li></ul></li></ol> 2. Isolement du muscle <ol><li> Dans un seau à glace, mettre trois béchers 10 ml, broyeurs tissus Potter Elvehjem et tous les autres instruments nécessaires / fournitures sur la glace. Tout doit rester glacée toute l&#39;expérience. </li><li> Placez IB1 et IB2 dans un seau à glace et EB dans le bain d&#39;eau chaude lorsque vous avez terminé. </li><li> Dans les trois béchers 10 ml, les solutions suivantes doivent être ajoutées: <ul><li> Bécher 1: 10 ml de PBS </li><li> Bécher 2: 10 ml de PBS / EDTA </li><li> Bécher 3: 3 ml de IB1 </li></ul></li><li> Tuer un rat humainement approuvé par votre soin des animaux et du Comité institutionnel local utilisation. </li><li> Isoler rapidement 250-500 mg de muscle squelettique, rincer dans le bécher 1, puis transfert à Bécher 2. </li></ol> 3. Homogénéisation / Isolement mitochondriale <ol><li> Transfert de muscle pour bécher de 3 et finement hachée le muscle avec des ciseaux. </li><li> Transférer la solution dans l&#39;homogénéisateur Potter-Elvehjem. Homogénéiser le muscle à l&#39;aide d&#39;un pilon motorisé (une perceuse va faire le travail) 10 fois gardant le tube dans la glace en tout temps. </li><li> Transfert homogénat de pré-réfrigérée microtubes de 2 ml et centrifuger à 700 g pendant 10 minutes à 4 ° C. </li><li> Transférer le surnageant dans un microtube de pré-réfrigérée nouvelle. Jeter granulés. </li><li> Centrifuger le surnageant au 10500 g pendant 10 minutes à 4 ° C. </li><li> Transférer le surnageant dans un microtube de pré-réfrigérée nouvelle et SN1 étiquette (numéro de surnageant 1) et le type de muscle. </li><li> Re-suspendre le culot dans 500 ul de IB2. </li><li> Centrifuger à 10500 g pendant 10 minutes à 4 ° C. </li><li> Transférer le surnageant dans un microtube de pré-réfrigérée nouvelle et SN2 étiquette (numéro de surnageant 2) et le type de muscle. </li><li> Suspendre finale mitochondriale culot dans 100 ul d&#39;IB2. </li><li> Re-spin suspension finale mitochondrial dans Minifuge pendant quelques secondes. S&#39;il ya une boulette, le transfert surnageant dans un microtube de pré-réfrigérée nouveau et jeter le culot. </li><li> Déterminer la concentration de protéines en utilisant le dosage de Bradford 8. </li></ol> 4. Étalonnage de l&#39;électrode REMARQUE: étalonnage de l&#39;électrode complète lors de l&#39;isolement des mitochondries. <ol><li> Ajouter 100 ml d&#39;eau distillée à 250 ml flacon. Agiter vigoureusement pendant 20 minutes pour équilibrer l&#39;eau avec du gaz dans l&#39;atmosphère. </li><li> Ajouter quelques gouttes de 50% de KCl à l&#39;électrode oxygraphe. </li><li> Placer un petit morceau de papier à rouler Rizla bleu sur le dessus de l&#39;électrode. </li><li> Placer un morceau de membrane en PTFE sur le dessus du papier à rouler. </li><li> Appliquer la bague intérieure en utilisant l&#39;applicateur anneau et ensuite placer la bague extérieure dans le sillon de l&#39;électrode. </li><li> Connectez-électrodes et montons le reste de l&#39;équipement: plus grand anneau de plastique de base, la chambre des mitochondries, et les tuyaux d&#39;eau. </li><li> Mettez les boîtes oxygraphe et lancer le logiciel oxygraphe. </li><li> Ajouter l&#39;eau équilibrée à la chambre des mitochondries. </li><li> Ajouter le barreau d&#39;agitation et tourner à une vitesse de 60. </li><li> Commencer une nouvelle expérience. </li><li> Cliquez sur l&#39;étalonnage et de calibration, puis en phase liquide pour l&#39;encadré 1. Changement de température à 37 ° C et cliquez sur &quot;OK&quot; deux fois. </li><li> Quand &quot;Override&quot; changements &quot;OK&quot; cliquer dessus et ouvrir le réservoir d&#39;azote. </li><li> Placez la pointe connectéau réservoir d&#39;azote dans la chambre de l&#39;oxygène et d&#39;établir à zéro. Cliquez sur &quot;OK&quot; quand il passe de &quot;neutralisation&quot;. </li><li> Aspirer l&#39;eau et ajouter 500 ul de tampon EB aux chambres mitochondriale. </li><li> Chambre d&#39;étanchéité avec le piston. </li><li> Si vous utilisez plusieurs boîtes, répétez les étapes 11 à 15 pour l&#39;étalonnage de l&#39;encadré 2. </li></ol> 5. Respiration mitochondriale <ol><li> Démarrer une nouvelle expérience; le laisser fonctionner pendant environ 1 minute pour stabiliser le signal. </li><li> Ajouter le volume nécessaire de la suspension mitochondriale (étape 3.11) pour 150 g dans chaque chambre, un événement marque, et le laisser tourner pendant 1 minute. </li><li> Ajouter 10 ul d&#39;eau tiède 250 mM glutamate mM/125 / malate pour une concentration finale de 5 mM mM/2.5. Mark et de laisser fonctionner pendant 1 minute. Ceci est défini comme l&#39;État 2. </li><li> Ajouter 7,5 ul de 10 mM d&#39;ADP pour une concentration finale de 150 uM, marque, et laissez fonctionner pendant 30 secondes. Ceci est défini comme l&#39;État 3. </li><li> Ajouter un autre ul 7.5 de 10 mM d&#39;ADP, marque, et laissez fonctionner pendant 1,5 minutes. </li><li> Ajouter 0,5 ul de froid 10 mM oligomycine pour une concentration finale de 1 uM, marque, et laisser courir pendant 3 minutes. Ceci est défini comme l&#39;État 4. </li><li> Ajouter 1 ul de froid 0,1 mM FCCP pour une concentration finale de 0,2 uM, marque, et laisser courir pendant 3 minutes. Ceci est défini comme l&#39;État 5. </li><li> Lorsque vous avez terminé, fin expérimenter et sauver. </li><li> Acquérir des taux de respiration en utilisant le logiciel oxygraphe. </li><li> Entrez le facteur de normalisation: si l&#39;ajout de 150 ug de protéine mitochondriale, le facteur de normalisation sera de 0,15. </li><li> Calculer le taux de respiration normalisé pour les états de respiration différente. </li><li> Calculer le rapport de contrôle respiratoire (RCR), en divisant l&#39;État par État 3 4. </li></ol> 6. Les résultats représentatifs: <img src="/files/ftp_upload/2452/2452fig1.jpg" alt="Figure 1" /> Figure 1. Montre un représentant de traçage de la consommation d&#39;oxygène par les muscles squelettiques mitochondries. Après le signal de l&#39;électrode est stabilisée, l&#39;échantillon mitochondries est ajouté à la chambre oxygraphe. Etat 2 la respiration commence avec l&#39;ajout de glutamate et le malate. Ajout d&#39;ADP augmente la consommation d&#39;oxygène, en définissant l&#39;état 3 respiration. ATP synthase est bloquée par addition de oligomycine pour obtenir l&#39;état 4 respiration. Enfin, FCCP est ajouté à découpler la respiration mitochondriale (état 5). Le tableau 1 montre les taux de consommation d&#39;oxygène représentant des États-2, 3, 4 et 5. Le ratio de contrôle respiratoire (RCR) est calculé pour chaque expérience. RCR ≥ 4 est considéré comme une preuve d&#39;une préparation viables mitochondries. <table border="1"><tbody><tr><td> Etats </td><td> Tarifs normalisé </td></tr><tr><td> État 2 </td><td> 34,74 </td></tr><tr><td> État 3 </td><td> 153,40 </td></tr><tr><td> État 4 </td><td> 16,49 </td></tr><tr><td> État 5 </td><td> 143,70 </td></tr><tr><td> RCR </td><td> 9,30 </td></tr></tbody></table> Tableau 1. Taux de respiration mitochondriale. Representativeoxygen taux de consommation du muscle squelettique mitochondries. Les valeurs sont normalisées à la quantité de mitochondries ajouté à la chambre. Le ratio de contrôle respiratoire (RCR) est déterminé en divisant l&#39;état 3 par l&#39;Etat 4. Un RCR ≥ 4 représente une préparation viables mitochondries.

<ol>
	<li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nat Protoc. 2, 287-295 (2007).</li><li>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=Roles+of+mitochondria+in+health+and+disease.">Roles of mitochondria in health and disease.</a> Diabetes. 53, S96-S102 (2004).</li><li>Johannsen, D. L., Ravussin, E. <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+mitochondria+in+health+and+disease.">The role of mitochondria in health and disease.</a> Curr Opin Pharmacol. , (2009).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 80, 3-44 (2007).</li><li>Pallotti, F., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+subfractionation+of+mitochondria+from+animal+cells+and+tissue+culture+lines.">Isolation and subfractionation of mitochondria from animal cells and tissue culture lines.</a> Methods Cell Biol. 65, 1-35 (2001).</li><li>Gamboa, J. L., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+content+and+distribution+changes+specific+to+mouse+diaphragm+after+chronic+normobaric+hypoxia.">Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia.</a> Am J Physiol Regul Integr Comp Physiol. , (2009).</li><li>Patel, S. P., Gamboa, J. L., McMullen, C. A., Rabchevsky, A., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lower+respiratory+capacity+in+extraocular+muscle+mitochondria:+evidence+for+intrinsic+differences+in+mitochondrial+composition+and+function.">Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function.</a> Invest Ophthalmol Vis Sci. 50, 180-186 (2009).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li></ol>

Nous présentons un protocole pour isoler les mitochondries viables à partir des muscles squelettiques. Si le rendement est un problème, le protocole peut être modifié en incubant le muscle isolé dans 5 ml de trypsine EDTA/0.01% PBS/10mM pendant 30 minutes dans la glace. Pour assurer la digestion complète du muscle à la trypsine, le muscle doit être pleinement hachée. Après l&#39;incubation de 30 minutes, la solution de trypsine PBS/10mM EDTA/0.01% doit être entièrement remplacé par 3 ml de tampon d&#39;iso...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>95% CO2 / 5% O2 mix</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Adenosine 5&#x2032;-diphosphate sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A2754</td>
<td>&nbsp;</td>
<tr>
<td>Blue Rizla Paper</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>890101</td>
<td>&nbsp;</td>
<tr>
<td>Bradford protein assay</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>500-0006</td>
<td>&nbsp;</td>
<tr>
<td>Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2920</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge 5804R</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Compressed nitrogen</td>
<td>&nbsp;</td>
<td>Local gas company</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>D-mannitol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9647</td>
<td>&nbsp;</td>
<tr>
<td>Ethlyene-glycol-bis-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E3889</td>
<td>&nbsp;</td>
<tr>
<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0728</td>
<td>&nbsp;</td>
<tr>
<td>Free fatty acid bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5889</td>
<td>&nbsp;</td>
<tr>
<td>HEPES sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7006</td>
<td>&nbsp;</td>
<tr>
<td>Isotemp 3006D</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>Male Sprague Dawley Rats</td>
<td>&nbsp;</td>
<td>Harlan</td>
<td>300-500g</td>
<td>&nbsp;</td>
<tr>
<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9138</td>
<td>&nbsp;</td>
<tr>
<td>Minifuge</td>
<td>&nbsp;</td>
<td>ISC Bioexpress</td>
<td>C1301P</td>
<td>&nbsp;</td>
<tr>
<td>Oligomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>O4876</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen electrode disc</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S1</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Oxygraph Plus V1.01 Software</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>pH-meter</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>1225506149</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Potassium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8416</td>
<td>&nbsp;</td>
<tr>
<td>Potter-Elvehjem homogenizers</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-414-14A</td>
<td>&nbsp;</td>
<tr>
<td>PTFE (0.0125mm &#215; 25mm) membrane</td>
<td>&nbsp;</td>
<td>Hansatech</td>
<td>S4</td>
<td>&nbsp;</td>
<tr>
<td>SKIL 3320 drill press</td>
<td>&nbsp;</td>
<td>Hardware store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5016</td>
<td>&nbsp;</td>		</tbody>
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mitochondrial isolation from skeletal muscle

Les mitochondries sont des organites le contrôle de la vie et la mort de la cellule. Ils participent à des réactions métaboliques clés, synthétiser plus de l&#39;ATP, et de réglementer un certain nombre de cascades de signalisation 2,3. Les chercheurs actuels et passés ont mitochondries isolées à partir des tissus de rats et de souris, comme le foie, le cerveau et le coeur 4,5. Ces dernières années, de nombreux chercheurs se sont concentrés sur l&#39;étude de la fonction mitochondriale des muscles squelettiques. Ici, nous décrivons une méthode que nous avons utilisé avec succès pour l&#39;isolement des mitochondries des muscles squelettiques 6. Notre procédure exige que tous les tampons et les réactifs sont frais et ont besoin d&#39;environ 250-500 mg de muscle squelettique. Nous avons étudié les mitochondries isolées de rat et de souris gastrocnémien et le diaphragme et les muscles extraoculaires chez le rat. Concentration de la protéine mitochondriale est mesurée avec le dosage de Bradford. Il est important que les échantillons mitochondriale être conservés glacée pendant la préparation et que des études fonctionnelles être effectuée dans un temps relativement court (~ 1 h). Respiration mitochondriale est mesurée en utilisant la polarographie avec une électrode de type Clark (système oxygraphe) à 37 ° C 7. Calibrage de l&#39;électrode à oxygène est une étape clé dans ce protocole et elle doit être effectuée quotidiennement. Mitochondries isolées (150 ug) sont ajoutés à 0,5 ml de tampon expérimental (EB). Etat 2 la respiration commence avec l&#39;ajout de glutamate (5mm) et malate (2,5 mM). Ensuite, l&#39;adénosine diphosphate (ADP) (150 uM) est ajouté pour commencer l&#39;état 3. Oligomycine (1 pM), un bloqueur synthase ATPase, est utilisé pour estimer l&#39;état 4. Enfin, le cyanure de p-carbonyle [trifluorométhoxy]-phényl-hydrazone (FCCP, 0,2 M) est ajouté à measurestate 5, ou la respiration découplée 6. Le ratio de contrôle respiratoire (RCR), le ratio de l&#39;état 3 à l&#39;état 4, est calculé après chaque expérience. Un RCR ≥ 4 est considéré comme une preuve d&#39;une préparation viables mitochondries. En résumé, nous présentons une méthode pour l&#39;isolement des mitochondries viables à partir des muscles squelettiques qui peuvent être utilisés dans biochimiques (par exemple une activité enzymatique, immunodétection, protéomique), des études et fonctionnels (respiration mitochondriale).

Watch this Scientific Journal Video about Mitochondrial Isolation from Skeletal Muscle at JoVE.com

Mitochondrial Isolation from Skeletal Muscle

Ce protocole décrit une procédure pour étudier la respiration des mitochondries isolées de muscles squelettiques. Cette méthode a été adaptée à partir Scorrano<em&gt; Et al.</em&gt; (2007). La procédure d&#39;isolement des mitochondries nécessite environ 2 heures. La respiration mitochondriale peut être complété en environ 1 heure.

Isolement mitochondriale du muscle squelettique

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

mitochondrial-isolation-from-skeletal-muscle

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28.8K vues.  University of Kentucky. Ce protocole décrit une procédure pour étudier la respiration des mitochondries isolées de muscles squelettiques. Cette méthode a été adaptée à partir Scorrano Et al. (2007). La procédure d'isolement des mitochondries nécessite environ 2 heures. La respiration mitochondriale peut être complété en environ 1 heure.

Vidéo: Mitochondrial Isolation from Skeletal Muscle

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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Biologie

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purification des progéniteurs du muscle squelettique

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Isolement des vaisseaux sanguins dérivés multipotentes précurseurs du muscle squelettique humain

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Isolement des mitochondries à partir des quantités minimes de souris muscle squelettique pour les mesures à haut débit microplaques respiratoires

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

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.

Isolement des mitochondries Intact du muscle squelettique par centrifugation différentielle pour respirométriques mesures haute résolution

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolement et caractérisation d’exosomes à partir de fibroblastes musculaires squelettiques

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Isolement des cardiomyocytes ventriculaires humains des tranches myocardales vibratoires-cut

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Isolement des mitochondries du muscle squelettique de la souris pour les tests respirométriques

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...