Questo lavoro è stato sostenuto da un finanziamento della National Eye Institute (R01 EY12998) per FH Andrade.

1. Preparazione del buffer <ol><li> Accendere 5804R centrifuga e impostare a 4 ° C. Accendere bagnomaria Isotemp 3006D e impostare a 37 ° C. </li><li> Preparare le seguenti soluzioni prima di isolamento del muscolo: <ul><li> PBS: sciogliere tampone fosfato (PBS) compresse in acqua distillata (5 compresse / litro). Mescolare bene. </li><li> PBS + 10 mM EDTA: Per preparare un 100 ml di soluzione, aggiungere 2 ml di 500 mM EDTA a 98 ml di PBS. </li><li> 8X Mitocondri buffer: 10,28 g di saccarosio per una concentrazione finale di 0,6 M, 400 mg di acidi grassi liberi albumina di siero bovino (BSA) per una concentrazione finale del 0,8%, 2,08 g di HEPES per una concentrazione finale di 160 mM, pH a 7,4 e QS a 50 ml con acqua distillata. </li><li> Buffer isolamento 1 (IB1): Aggiungere 200 ml di 500 mM EDTA per una concentrazione finale di 10 mm, 0,392 g di D-mannitolo per una concentrazione finale di 215 mm, 1,25 ml di 8X mitocondri, pH a 7,4 e QS a 10 ml con acqua distillata. </li><li> Buffer isolamento 2 (IB2): Aggiungere 60 ml di 500 mM EGTA per una concentrazione finale di 3 mm, 0,392 g di D-mannitolo per una concentrazione finale di 215 mm, 1,25 ml di 8X mitocondri, pH a 7,4 e QS a 10 ml con acqua distillata. </li><li> Buffer sperimentale (EB): Aggiungere 100 ml di 500 mM MgCl 2 per una concentrazione finale di 5 mm, 0,392 g di D-mannitolo per una concentrazione finale di 215 mm, 25 ml di 2,5 mM KH 2 PO 4 per una concentrazione finale di 6,25 mM, 2 l di mM EGTA 100 per una concentrazione finale di 20 micron, 1,25 ml di tampone mitocondriale, pH a 7,4 e QS a 10 ml con acqua distillata. </li></ul></li></ol> 2. Isolamento muscolare <ol><li> In un secchio di ghiaccio, mettere tre bicchieri 10 ml, Potter-Elvehjem macinatori tessuto e tutti i necessari altri strumenti / supplies sul ghiaccio. Tutto deve rimanere ghiacciato per tutto l&#39;esperimento. </li><li> Luogo IB1 e IB2 nel secchiello del ghiaccio e EB in bagno d&#39;acqua calda quando hai finito. </li><li> Nei tre bicchieri 10 ml, le seguenti soluzioni si aggiungono: <ul><li> Bicchiere 1: 10 ml di PBS </li><li> Beaker 2: 10 ml di PBS / EDTA </li><li> Becher da 3: 3 ml di IB1 </li></ul></li><li> Uccidere un topo umanamente come approvato dal locale Cura degli animali e del Comitato Istituzionale Usa. </li><li> Isolare rapidamente 250-500 mg di muscolo scheletrico, sciacquare in Beaker 1, poi il trasferimento a Beaker 2. </li></ol> 3. Omogeneizzazione / isolamento mitocondriale <ol><li> Trasferimento muscolo Beaker 3 e tritare finemente il muscolo con le forbici. </li><li> Trasferire la soluzione del Potter-Elvehjem omogeneizzatore. Omogeneizzare il muscolo con un pestello motorizzata (un trapano a colonna farà il lavoro) 10 volte mantenendo il tubo in ghiaccio in ogni momento. </li><li> Trasferimento omogenato di provette precedentemente congelate microcentrifuga da 2 ml e centrifugare a 700 g per 10 minuti a 4 ° C. </li><li> Trasferire il supernatante in un nuovo pre-raffreddata provetta. Scartare pellet. </li><li> Centrifugare surnatante a 10.500 g per 10 minuti a 4 ° C. </li><li> Trasferire il supernatante in un nuovo pre-raffreddata provetta e SN1 etichetta (surnatante numero 1) e tipo muscolare. </li><li> Risospendere pellet in 500 ml di IB2. </li><li> Centrifugare a 10.500 g per 10 minuti a 4 ° C. </li><li> Trasferire il supernatante in un nuovo pre-raffreddata provetta e SN2 etichetta (surnatante numero 2) e di tipo muscolare. </li><li> Sospendere finale mitocondriale pellet in 100 ml di IB2. </li><li> Re-spin sospensione finale mitocondriale in minifuge per alcuni secondi. Se c&#39;è una pallina, trasferire il supernatante in un nuovo pre-raffreddata provetta e gettare il pellet. </li><li> Determinare la concentrazione di proteine ​​con il saggio Bradford 8. </li></ol> 4. Elettrodo di calibrazione NOTA: calibrazione dell&#39;elettrodo Completa durante l&#39;isolamento mitocondriale. <ol><li> Aggiungere 100 ml di acqua distillata matraccio da 250 ml. Agitare vigorosamente per 20 minuti per equilibrare l&#39;acqua con gas atmosferici. </li><li> Aggiungere qualche goccia di 50% KCl all&#39;elettrodo Oxygraph. </li><li> Posizionare un piccolo pezzo di carta blu Rizla rotolamento sopra di elettrodo. </li><li> Posizionare un pezzo di membrana in PTFE sulla parte superiore del foglio di rotolamento. </li><li> Applicare l&#39;anello interno con l&#39;applicatore anello e poi posto l&#39;anello esterno nella scanalatura elettrodo. </li><li> Collegare gli elettrodi e assemblare il resto delle attrezzature: grande base del cilindro di plastica, camera mitocondriale, e tubi dell&#39;acqua. </li><li> Accendere scatole Oxygraph e avviare il software Oxygraph. </li><li> Aggiungere l&#39;acqua equilibrati a camera mitocondriale. </li><li> Aggiungi mescolare bar e girare a una velocità di 60. </li><li> Iniziare un nuovo esperimento. </li><li> Clicca su calibrare e poi calibrazione fase liquida per Box 1. Variazione di temperatura a 37 ° C e fare clic su &quot;OK&quot; due volte. </li><li> Quando &quot;override&quot; diventa &quot;OK&quot; clic su di esso e aprire il serbatoio di azoto. </li><li> Luogo punta collegatoal serbatoio di azoto in camera e stabilire ossigeno pari a zero. Fare clic su &quot;OK&quot; quando si passa da &quot;Sostituisci&quot;. </li><li> Aspirare l&#39;acqua e aggiungere 500 microlitri di tampone EB alle camere mitocondriale. </li><li> Camera di tenuta con stantuffo. </li><li> Se si utilizzano più caselle, ripetere i passaggi 11-15 per la calibrazione del riquadro 2. </li></ol> 5. Respirazione mitocondriale <ol><li> Inizia un nuovo esperimento, farlo funzionare per circa 1 minuto per stabilizzare il segnale. </li><li> Aggiungere il volume necessario di sospensione mitocondriale (passo 3.11) per 150 mg in ogni camera, evento marchio, e farlo funzionare per 1 minuto. </li><li> Aggiungere 10 ml di caldo 250 mM/125 mM glutammato / malato per una concentrazione finale di 5 mM mM/2.5. Mark e lasciare correre per 1 minuto. Questo è definito come Stato 2. </li><li> Aggiungere 7,5 ml di ADP 10 mm per una concentrazione finale di 150 micron, segno, e lasciare per 30 secondi. Questo è definito come Stato 3. </li><li> Aggiungere altri 7,5 ml di 10 mM ADP, marchio, e lasciare correre per 1,5 minuti. </li><li> Aggiungere 0,5 l di freddo oligomicina 10 mm per una concentrazione finale di 1 micron, segno, e lasciare correre per 3 minuti. Questo è definito come Stato 4. </li><li> Aggiungere 1 ml di mM FCCP freddo 0.1 per una concentrazione finale di 0,2 micron, segno, e lasciare correre per 3 minuti. Questo è definito come Stato 5. </li><li> Al termine, fine sperimentare e salvare. </li><li> Acquisire i tassi di respirazione utilizzando il software Oxygraph. </li><li> Inserisci il fattore di normalizzazione: se l&#39;aggiunta di 150 mcg di proteine ​​mitocondriali, il fattore di normalizzazione sarà 0,15. </li><li> Calcolare i tassi di respirazione normalizzata per la respirazione stati diversi. </li><li> Calcolare il rapporto di controllo respiratorio (RCR) dividendo Stato 3 per Stato 4. </li></ol> 6. Rappresentante dei risultati: <img src="/files/ftp_upload/2452/2452fig1.jpg" alt="Figura 1" /> Figura 1. Mostra un rappresentante tracciamento del consumo di ossigeno da parte dei muscoli scheletrici mitocondri. Dopo il segnale dell&#39;elettrodo è stabilizzato, il campione mitocondri viene aggiunta alla camera Oxygraph. Stato 2 respirazione inizia con aggiunta di glutammato e malato. L&#39;aggiunta di ADP aumenta il consumo di ossigeno, la definizione di stato 3 respirazione. ATP sintasi è bloccato con l&#39;aggiunta di oligomicina ottenere Stato 4 della respirazione. Infine, FCCP viene aggiunto sganciare la respirazione mitocondriale (stato 5). Tabella 1 mostra i tassi di consumo di ossigeno di rappresentanza per gli stati 2, 3, 4 e 5. Il rapporto di controllo respiratorio (RCR) è calcolato per ogni esperimento. RCR ≥ 4 è considerata come prova di una preparazione valida mitocondri. <table border="1"><tbody><tr><td> Stati </td><td> Tariffe normalizzato </td></tr><tr><td> Stato 2 </td><td> 34,74 </td></tr><tr><td> Stato 3 </td><td> 153,40 </td></tr><tr><td> Stato 4 </td><td> 16,49 </td></tr><tr><td> Stato 5 </td><td> 143,70 </td></tr><tr><td> RCR </td><td> 9,30 </td></tr></tbody></table> Tabella 1. Tassi di respirazione mitocondriale. Tassi di consumo Representativeoxygen dal muscolo scheletrico mitocondri. I valori sono normalizzati alla quantità di mitocondri aggiunta alla camera. Il rapporto di controllo respiratorio (RCR) è determinato dividendo stato 3 per stato 4. Un RCR ≥ 4 rappresenta una valida preparazione mitocondri.

<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>

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Vi presentiamo un protocollo per isolare i mitocondri praticabile da muscoli scheletrici. Se il rendimento è un problema, il protocollo può essere modificato tramite l&#39;incubazione del muscolo isolato in 5 ml di tripsina PBS/10mM EDTA/0.01% per 30 minuti in ghiaccio. Per assicurare la completa digestione con tripsina muscolare, il muscolo ha bisogno di essere completamente tritato. Dopo i 30 minuti di incubazione, la soluzione PBS/10mM tripsina EDTA/0.01% deve essere completamente sostituita con 3 ml di buffer di i...

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<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>
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<td>Centrifuge 5804R</td>
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<td>Eppendorf</td>
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<td>Compressed nitrogen</td>
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<td>Local gas company</td>
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<td>D-mannitol</td>
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<td>Sigma</td>
<td>M9647</td>
<td>&nbsp;</td>
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<td>Ethlyene-glycol-bis-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E3889</td>
<td>&nbsp;</td>
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<td>Ethylenediaminetetraacetic acid (EDTA)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0728</td>
<td>&nbsp;</td>
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<td>Free fatty acid bovine serum albumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A8806</td>
<td>&nbsp;</td>
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<td>Glutamic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5889</td>
<td>&nbsp;</td>
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<td>HEPES sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H7006</td>
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<td>Isotemp 3006D</td>
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<td>Fisher Scientific</td>
<td>&nbsp;</td>
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<td>Magnesium chloride</td>
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<td>Sigma</td>
<td>M8266</td>
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<td>Male Sprague Dawley Rats</td>
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<td>Harlan</td>
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<td>Malic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M9138</td>
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<td>C1301P</td>
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<td>O4876</td>
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<td>Hansatech</td>
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<td>Oxygraph Plus V1.01 Software</td>
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<td>Hansatech</td>
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<td>Mettler Toledo</td>
<td>1225506149</td>
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<td>Phosphate-buffered saline (PBS)</td>
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<td>Sigma</td>
<td>P4417</td>
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<td>Potassium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P3911</td>
<td>&nbsp;</td>
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<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>
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<td>SKIL 3320 drill press</td>
<td>&nbsp;</td>
<td>Hardware store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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mitochondrial isolation from skeletal muscle

I mitocondri sono organelli controllare la vita e la morte della cellula. Partecipano in chiave reazioni metaboliche, sintetizzare la maggior parte di ATP, e regolano una serie di cascate di segnalazione 2,3. Ricercatori passati e presenti hanno mitocondri isolati da tessuti ratti e topi, come fegato, cervello e cuore 4,5. Negli ultimi anni, molti ricercatori si sono concentrati sullo studio di funzione mitocondriale dei muscoli scheletrici. Qui, descriviamo un metodo che abbiamo usato con successo per l&#39;isolamento dei mitocondri da muscoli scheletrici 6. La nostra procedura richiede che tutti i buffer e reagenti sono freschi e hanno bisogno di circa 250-500 mg di muscolo scheletrico. Sono stati studiati i mitocondri isolati da gastrocnemio ratto e nel topo e il diaframma e muscoli extraoculari ratto. Concentrazione di proteina mitocondriale viene misurata con il saggio Bradford. E &#39;importante che i campioni mitocondriale essere tenuti ghiacciata durante la preparazione e che gli studi funzionali da eseguire in un tempo relativamente breve (~ 1 ora). Respirazione mitocondriale è misurata con polarografia con un elettrodo di Clark-tipo (sistema Oxygraph) a 37 ° C 7. Taratura degli elettrodi ossigeno è un passo fondamentale di questo protocollo e deve essere effettuata ogni giorno. Mitocondri isolati (150 mcg) vengono aggiunti 0,5 ml di tampone sperimentale (EB). Stato 2 respirazione inizia con aggiunta di glutammato (5mm) e malato (2,5 mm). Poi, adenosina difosfato (ADP) (150 mM) è stato aggiunto a iniziare 3. Oligomicina (1 micron), un bloccante sintasi ATPasi, viene utilizzato per stimare lo stato 4. Infine, carbonile cianuro p-[trifluoromethoxy]-fenil-idrazone (FCCP, 0,2 mM) si aggiunge a measurestate 5, o sganciati respirazione 6. Il rapporto di controllo respiratorio (RCR), il rapporto tra stato 3 allo stato 4, viene calcolato dopo ogni esperimento. Un RCR ≥ 4 è considerata come prova di una preparazione valida mitocondri. In sintesi, vi presentiamo un metodo per l&#39;isolamento dei mitocondri vitali da muscoli scheletrici che può essere utilizzato in biochimica (ad esempio, l&#39;attività enzimatica, immunolocalizzazione, proteomica) e studi funzionali (respirazione mitocondriale).

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

Mitochondrial Isolation from Skeletal Muscle

Questo protocollo descrive una procedura per studiare la respirazione dei mitocondri isolati da muscolo scheletrico. Questo metodo è stato adattato da Scorrano<em&gt; Et al.</em&gt; (2007). La procedura di isolamento dei mitocondri richiede circa 2 ore. La respirazione mitocondriale può essere completata in circa 1 ora.

Isolamento dei mitocondri dal muscolo scheletrico

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

Research

JoVE Journal

Biology

28.8K Views.  University of Kentucky. Questo protocollo descrive una procedura per studiare la respirazione dei mitocondri isolati da muscolo scheletrico. Questo metodo è stato adattato da Scorrano Et al. (2007). La procedura di isolamento dei mitocondri richiede circa 2 ore. La respirazione mitocondriale può essere completata in circa 1 ora.

Video: 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.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

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

Ricerca

Biologia

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.

Purificazione di progenitori dal muscolo scheletrico

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.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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.

Isolamento dei vasi sanguigni derivati ​​da Multipotent precursori del muscolo scheletrico umano

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.

L&#39;isolamento di mitocondri da minime quantità di muscolo scheletrico del mouse per misurazioni High Throughput micropiastre respiratorie

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.

L&#39;isolamento di mitocondri intatti dal muscolo scheletrico mediante centrifugazione differenziale per alta risoluzione respirometria Misure

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.

Isolamento e caratterizzazione di esosomi da fibroblasti muscolari scheletrici

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.

Isolamento dei cardiomiociti ventricolari umani dalle fette miocardici Vibratome-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.

Isolamento dei mitocondri dal muscolo scheletrico del topo per saggi respirometrici

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 ...

Decellularization of Skeletal Muscle 

To begin, inspect the muscles of interest dissected from the sacrificed mouse for the absence of bone chips or ragged edges and trim them away with sharp scissors if needed. Weigh the cleaned muscles using an analytical balance and record their weight. After placing the muscle in at least 0.1 milliliters of 1%SDS per milligram of weight, place the well plate on a rocking shaker set to 50 to 80 Hertz.Visually inspect the SDS solution periodically. When the solution becomes cloudy, remove the solution with a pipette without aspirating the muscles and replace it with an equal volume of fresh 1%SDS. When the solution remains clear for 24 hours, remove the final SDS solution from the decellularized muscles and replace it with an equal volume of PBS.Inspect the decellularized muscles under a stereo microscope and carefully remove any hair or debris stuck to the muscle using forceps. Carefully remove the PBS and replace it with an equal volume of 3.7%formaldehyde before returning the plate to the rocking shaker for 24 hours.