1. Tessuto Collezione: <ol><li> Utilizzare topi tra 7 e 12 settimane di età. Di solito, almeno due animali sono utilizzati: uno è quello di impostare il FACS monocolore controlli necessari per il risarcimento e per campioni isotipo fluorescenza-meno-un controllo. L&#39;altro è il campione reale per l&#39;ordinamento. </li><li> Euthanize i topi da CO 2 inalazione o secondo un protocollo di etica della scelta. Topi posto su un tovagliolo di carta, rivolta verso l&#39;alto, e spruzzare con il 70% di etanolo a fondo per evitare la contaminazione con la pelliccia del mouse. </li><li> Sollevare la cute nella regione addominale con una pinza, poi fare un piccolo taglio longitudinale con forbici affilate. </li><li> Sbucciate i due lembi di pelle in direzioni opposte (su e giù) per esporre completamente i muscoli degli arti posteriori sotto. </li><li> Dell&#39;accisa il tessuto muscolare inserendo ed estendendo le forbici lungo entrambi i lati della tibia. </li><li> Ripetere la stessa procedura per il femore per estrarre il tessuto muscolare. </li><li> Mettere tutti i tessuti muscolari asportati in 60 millimetri di Petri-piatti, utilizzando un piatto per il tessuto di ogni topo. </li><li> Ripetere quanto sopra per tutti i mouse. </li></ol> 2. Dissociare tessuto in cellule singole per digestione enzimatica: In questa sezione del protocollo, utilizzare i reagenti e strumenti sterili e lavorare in condizioni sterili. <ol start="9"><li> Aggiungere 2 ml di Collagenasi II (Sigma, cat # 6885, 500U / mL) e 20 l di 250mm CaCl 2 magazzino ad ogni Petri-piatto. </li><li> Lacerare il tessuto muscolare in piccoli pezzi (circa 1 mm 3) con due pinze (una seghettato, una con 2 denti, da Strumenti Scienze Arti, cat # 11051-10, 11154-10), facendo attenzione a rimuovere ed eliminare il più contaminati non-tessuto muscolare possibile. </li><li> Coprire il Petri-piatti e incubare loro a 37 gradi centigradi per 30 minuti. </li><li> Recuperare il Petri-piatti e usare una siringa da 3 ml stantuffo a schiacciare a fondo i pezzi di tessuto. Utilizzare uno stantuffo per ogni campione per evitare contaminazioni. </li><li> Aggiungete un po &#39;(~ 5 ml) PBS freddo sterile ad ogni Petri-piatto e trasferire i fanghi tessuto in un tubo 50mL Falcon (sciacquare la Petri-piatto, se necessario, di recuperare tutti i tessuti) </li><li> Agitare la provetta Falcon vigorosamente, riempire la provetta con PBS, centrifugare @ 800 giri x 5 minuti (da banco Eppendorf top Modello: 5810R), e aspirare il surnatante (ripetere 3 volte) </li><li> Aggiungere 1 ml di miscela Collagenasi D (Roche, Cat # 11088882001, 1,5 U / mL) / dispasi II (Roche, Cat #: 04942078001, 2.4u / mL) e 10μL di CaCl 2 (250mm) a ciascuna provetta, incubare a 37 gradi Celsius con rotazione per un&#39;ora. </li><li> Aggiungere 5-10 ml di freddo, sterile PBS, vortice e pipetta accuratamente per omogeneizzare il tessuto. </li><li> Riempire i tubi con PBS e mescolare bene. </li><li> Trasferire il liquido in un colino cellula 40μm collocato su un tubo 50 ml Falcon per filtrare i pezzi di altri grandi produttori di tessuto. Dividere ogni campione uniformemente in tre 50mL tubi Falcon. Riempire ciascuno dei tubi con PBS quindi si centrifuga @ 1600rpm per 5 minuti a 8 gradi Celsius. </li><li> Eliminare il supernatante e raccogliere le cellule per la colorazione. </li></ol> 3. Anticorpi colorazione e ordinamento: In questa sezione del protocollo, l&#39;uso FACS tamponi sterili e la raccolta e il lavoro in condizioni sterili. Anticorpi commerciali sono generalmente considerati sterili se correttamente gestita. <ol start="20"><li> Per singolo colore e campioni di controllo isotipico, risospendere il campione in 1 ml di buffer FACS e dividere le cellule sospese in nove 1,5 ml di pre-etichettati tubi Eppendorf (100 l ogni tubo). <ol><li> Impostare colorazione singolo colore si mescola in base alla seguente tabella: ATTENZIONE: La concentrazione finale di anticorpi isotipo di controllo nella macchia dovrebbe corrispondere a quello dell&#39;anticorpo primario sono il controllo. Per esempio, se anti-Sca-1 è usato per 1 μg/10 6 celle, il suo isotipo corrispondente deve essere utilizzato alla stessa concentrazione. <table border="1"><tbody><tr><td> Tubo # </td><td> Nome del tubo </td><td> Azienda </td><td> Cat # </td><td> clone </td><td> isotipo </td><td> diluizione </td></tr><tr><td> 1 </td><td> Non macchia </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2 </td><td> Hoechst33342 </td><td> sigma </td><td> B2261 </td><td></td><td></td><td> 2-5ug/ml </td></tr><tr><td> 3 </td><td> PI </td><td> sigma </td><td> P4864 </td><td></td><td></td><td> 1ug/ml </td></tr><tr><td rowspan="2"> 4 </td><td rowspan="2"> CD31-FITC CD45-FITC </td><td> eBioscience </td><td> 11-0311-85 </td><td> 390 </td><td> Rat IgG2a </td><td> 500 </td></tr><tr><td> Fatti in casa </td><td></td><td> I3 / 2 </td><td> Rat IgG2b </td><td> 500 </td></tr><tr><td> 5 </td><td> SCA1-PECY7 </td><td> eBioscience </td><td> 25-5981-82 </td><td> D7 </td><td> Rat IgG2a </td><td> 5000 </td></tr><tr><td> 6 </td><td> α-7 APC </td><td> Fatti in casa </td><td></td><td> R2F2 </td><td> Rat IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Impostare colorazione controllo isotipico mescola con la seguente tabella: <table border="1"><tbody><tr><td> Tubo # </td><td> Nome di tubo </td><td> Hoechst </td><td> PI </td><td> CD31- FITC </td><td> CD45- FITC </td><td> Scal- PE-Cy7 </td><td> A-7- APC </td><td> Rat- IgG2b- APC </td><td> Rat- IgG2a- pecy7 </td><td> Rat- IgG2a- FITC </td><td> Rat- IgG2b- FITC </td></tr><tr><td> 7 </td><td> Iso- CD31 / CD45 </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td><td> - </td><td> - </td><td> + </td><td> + </td></tr><tr><td> 8 </td><td> Iso- Scal- pecy7 </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td></tr><tr><td> 9 </td><td> Iso- α-7-APC </td><td> + </td><td> + </td><td> + </td><td> + </td><td> + </td><td> - </td><td> + </td><td> - </td><td> - </td><td> - </td></tr></tbody></table></li><li> Pipettare il singolo colore e di isotopi mescola colorazione di controllo (di solito regolare la concentrazione di anticorpi in modo che tra 5 e 10 ml di colorazione miscela vengono aggiunti ad ogni campione) nel tubo Eppendorf appropriato dal punto 20. Mescolare bene e incubare in ghiaccio per 1 ora. </li></ol></li><li> Macchia cellule di topo campione con 800μL del cocktail di anticorpi (in basso) per l&#39;ordinamento delle cellule FACS. Mescolare bene e incubare in ghiaccio per 1 ora. Anticorpi cocktail: Hoechst, PI, CD31-FITC, CD45-FITC, SCA1-PECY7, α-7 APC ATTENZIONE: Il rapporto ottimale di anticorpi contro le cellule devono essere determinata individualmente ogni titolazione degli anticorpi, in quanto possono esistere differenze significative tra i lotti. La quantità ottimale di anticorpi da utilizzare devono essere espressi in μgs di anticorpi per 10 6 cellule bersaglio e mantenuta costante durante l&#39;utilizzo dello stesso lotto di anticorpi. Chiaramente, se il numero di cellule bersaglio non cambia significativamente tra gli esperimenti, o se il volume colorazione è in scala con il numero di cellule, una diluizione fisso di ciascun anticorpo può essere utilizzato anche. </li><li> Lavaggio: Aggiungere circa 1 ml di buffer di FACS per ognuna delle 9 provette di controllo, aggiungere circa 20 mL di tampone FACS alla provetta. </li><li> Centrifugare le provette Eppendorf @ 3000rpm per 5 minuti (da banco centrifuga Eppendorf in alto, Modello: 5417C). Centrifugare la provetta @ 1600rpm per 5 minuti (da banco centrifuga Eppendorf in alto, Modello: 5810R). </li><li> Aspirare e scartare il supernatante da tutti i tubi. </li><li> Risospendere le cellule nel buffer FACS (1 ml per ciascuno dei controlli, da 4 ml per il campione ordinamento), le cellule Pipettare in &quot;tubi FACS&quot; attraverso il filtro delle cellule tappo (cat BD Falcon # 352235, 40μm dimensioni dei pori) per rimuovere macchie rimanenti che possono influire sul citometro. </li><li> Impostare il FACS sorter con controlli singolo colore e controlli isotipo. </li><li> Raccogliere le cellule ordinati secondo la tabella seguente in un buffer di 3,5 ml di raccolta (95% DMEM, 5% FBS) separatamente. Normalmente, quando la raccolta entrambi gli arti posteriori, un mouse di tipo selvatico potrebbe produrre circa 150.000 cellule progenitrici miogeniche o 100.000 cellule progenitrici adipogenico, con la vitalità superiore al 95%, come giudicato da rianalizzati cellule ordinati per FACS al termine della procedura (Figura 1 ) Definizione dei progenitori miogenici e adipogenico base alla colorazione della superficie <table border="1"><tbody><tr><td> Anticorpo / macchia </td><td> Progenitrici miogenici (MP) </td><td> Progenitrici adipogenico (AP) </td></tr><tr><td> Hoechst </td><td> + </td><td> + </td></tr><tr><td> PI </td><td> - </td><td> - </td></tr><tr><td> CD31-FITC </td><td> - </td><td> - </td></tr><tr><td> CD45-FITC </td><td&gt; - </td><td> - </td></tr><tr><td> SCA1-PECY7 </td><td> - </td><td> + </td></tr><tr><td> α-7 APC </td><td> + </td><td> - </td></tr></tbody></table></li></ol> 4. Trapianto: <ol start="28"><li> Centrifugare le cellule ordinati @ 1500rpm per 5 minuti, togliere le cellule surnatante e trasferimento in autoclave i tubi Eppendorf (1,5 ml), lavare le cellule con 500 l di PBS sterile (i livelli di free!), Quindi si centrifuga @ 3000rpm per 5 minuti. Il mantenimento di tutti i reagenti in condizioni sterili e lavorare in un cappuccio colture di tessuti, risospendere progenitori miogenici in PBS, o progenitrici adipogenico in Matrigel (BD cat # 354234) a circa 10 6 cellule / ml. </li><li> Trapianto di progenitori miogenici o progenitrici adipogenico ai topi destinatario. <ol><li> Anestetizzare il mouse con isoflouran secondo la vostra politica istituzione. </li><li> Radersi i capelli intorno alla regione di iniezione, poi sterilizzare la pelle strofinando con etanolo al 70% </li><li> Iniettare 20μL dei progenitori miogenici o progenitori adipogenico (= 20k cellule) con un 3 / 10 siringa da insulina CC (cat BD # 309300). </li></ol></li><li> Dopo un tempo adeguato (tre settimane funziona bene, ma miofibre transgenici che esprimono i marcatori del donatore-derivati ​​sono di solito evidenti entro una settimana dopo il trapianto), anestetizzare i topi destinatario da passaggio 29 mediante iniezione intraperitoneale di 400 microlitri Avertin (Cat Sigma # T04840-2, 25 mg / ml), poi profumato transcardially con 50mL PBS (contiene 10 mM di EDTA) per primo, seguito da 15 ml del 4% PFA. Raccogliere tessuto bersaglio, post-fix con 4% PFA durante la notte, se necessario, poi il trasferimento al 20% di saccarosio durante la notte. Incorpora in ottobre, congelare e cryosection. </li></ol> 5. Rappresentante dei risultati: MP quando vengono trapiantate nel muscolo scheletrico del mouse, che prontamente si fondono con pre-esistente miofibre. Pertanto, qualsiasi etichetta genetico presente nelle cellule del donatore sarà facilmente individuabile nelle fibre che li ha accolti. La figura 2 mostra un esempio in cui le cellule trapiantate espresso umane phostphatase alcalino, ha rivelato istochimicamente. AP quando vengono trapiantate il risultato dipende in larga misura l&#39;ambiente del sito trapianto: quando queste cellule trapiantate per via sottocutanea dare origini a adipociti e miofibroblasti (vedi figura 2). In molti altri siti, compresi i muscoli, non sopravvivono a meno degenerazione grassa è stata indotta 3. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Figura 1" /> Figura 1. Ordinamento strategia per l&#39;isolamento delle popolazioni progenitrici dal muscolo scheletrico (A) strategia Ordinamento:. Cellule vitali sono state identificate sulla base di dispersione in avanti e side scatter. Colorazione Hoechst è stato utilizzato per escludere detriti anuclear e ioduro di propidio (PI) colorazione di escludere le cellule morte. Ematopoietiche (CD45) ed endoteliali (CD31), le cellule sono state escluse dalle porte di smistamento. Il α7 + Hoechst + PI - CD45 - CD31 - Scal - sottoinsieme contiene tutti i progenitori miogenici (MP). Il Scal + Hoechst + PI - CD45 - CD31 - α7 - popolazione contiene progenitori adipogenico (AP). (B) Fluorescenza-minus-one (FMO) controlla isotipo confermare la specificità della macchia. (C) Purezza controlli di sottoinsiemi MP e AP dopo l&#39;ordinamento. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Figura 2" /> Figura 2. Risultati rappresentativi seguenti MP e AP di trapianto. AP sono stati trapiantati per via sottocutanea (pannello superiore) e MP per via intramuscolare (pannello inferiore). In entrambi i casi, le cellule del donatore origine da un topo transgenico che esprime fosfatasi alcalina umano, identificato dalla colorazione marrone.

<ol>
	<li>Buckingham, M., Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+stem+cells.">Skeletal muscle stem cells.</a> Curr Opin Genet Dev. 18, 330-336 (2008).</li><li>Relaix, F., Marcelle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells.">Muscle stem cells.</a> Curr Opin Cell Biol. 21, 748-753 (2009).</li><li>Joe, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+injury+activates+resident+fibro/adipogenic+progenitors+that+facilitate+myogenesis.">Muscle injury activates resident fibro/adipogenic progenitors that facilitate myogenesis.</a> Nat Cell Biol. 12, 153-163 (2010).</li><li>Natarajan, A., Lemos, D. R., Rossi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibro/adipogenic+progenitors:+A+double-edged+sword+in+skeletal+muscle+regeneration.">Fibro/adipogenic progenitors: A double-edged sword in skeletal muscle regeneration.</a> Cell Cycle. 9, (2010).</li><li>Sherwood, R. I. <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+adult+mouse+myogenic+progenitors:+functional+heterogeneity+of+cells+within+and+engrafting+skeletal+muscle.">Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle.</a> Cell. 119, 543-554 (2004).</li><li>Sacco, A., Doyonnas, R., Kraft, P., Vitorovic, S., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-renewal+and+expansion+of+single+transplanted+muscle+stem+cells.">Self-renewal and expansion of single transplanted muscle stem cells.</a> Nature. , (2008).</li><li>Montarras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+isolation+of+satellite+cells+for+skeletal+muscle+regeneration.">Direct isolation of satellite cells for skeletal muscle regeneration.</a> Science. 309, 2064-2067 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

L&#39;obiettivo di questo protocollo è quello di trovare un equilibrio ragionevole tra alti rendimenti e alti vitalità delle cellule purificate. La fase più critica per garantire che le cellule sane sono state recuperate è, prevedibilmente, la dissociazione enzimatica del tessuto di partenza. Il trattamento del tessuto deve essere particolarmente delicato, che è difficile da dimostrare o apprezzare anche in un formato audiovisivo. Un altro fattore chiave è la durata della procedura. Più a lungo ci vuole per andare dal donatore al ricevente l&#39;animale, minore è la vitalità e, quindi, l&#39;efficienza attecchimento. In caso di domanda sulla vitalità che non è immediatamente risposto, cercando alla frequenza di eventi PI positivi nei campioni di cellule purificate dopo l&#39;ordinamento, si consiglia un test di diluizione limita a misurare clonogenicità viene eseguita 3. Sorta tipici muscolo danneggiato regolarmente resa di circa 1 in 15-20 cellule in grado di avviare colonie miogenici o fibro / adipogenico in vitro. Mentre praticamente del 100% delle colonie avviato da parlamentari contenere miotubi differenziati, solo circa un terzo delle colonie ottenute da FAP contenere adipociti oltre a miofibroblasti actina del muscolo liscio positivi.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Collagenasi II </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 U / ml </td></tr><tr><td> Collagenasi D </td><td> Roche </td><td> 11088882001 </td><td> 1,5 U / ml </td></tr><tr><td> Spostare II </td><td> Roche </td><td> 04942078001 </td><td> 2,4 u / ml </td></tr><tr><td> cellula filtro </td><td> BD </td><td> 352340 </td><td> 40 micron </td></tr><tr><td> Tubo con filtro cellulare </td><td> BD </td><td> 352235 </td><td> 40 micron </td></tr><tr><td> Hoechst33342 </td><td> Sigma-Aldrich </td><td> B2261 </td><td> 2-5 ug / ml </td></tr><tr><td> CD31-FITC </td><td> Ebiosciences </td><td> 11-0311-85 </td><td> Clone 390 </td></tr><tr><td> CD45-FITC </td><td> Fatto in casa </td><td></td><td> Clone I3 / 2 </td></tr><tr><td> SCA1-PECY7 </td><td> Ebiosciences </td><td> 25-5981-82 </td><td> Clone D7 </td></tr><tr><td> α-7 integrina APC </td><td> Fatto in casa </td><td> Informarsi con Rossi laboratorio </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

Muscolo scheletrico contiene popolazioni progenitore più di distinte origini embrionale e potenziale di sviluppo. Progenitori miogenici, di solito risiede in una &quot;posizione delle cellule satellite&quot; tra la membrana plasmatica miofibre e la laminina-ricchi membrana basale che ensheaths esso, sono auto-rinnovamento delle cellule che sono esclusivamente impegnati nel 1,2 lignaggio miogenico. Recentemente abbiamo descritto una seconda classe di progenitori nave associate in grado di generare e miofibroblasti adipociti bianchi, che risponde ai danni da entrare in modo efficace la proliferazione e fornisce supporto trofico per le cellule miogenici durante la rigenerazione dei tessuti 3,4. Uno dei test più affidabile per determinare il potenziale di sviluppo e rigenerazione di una popolazione di cellule dato si basa sul loro peso e il trapianto di 5-7. A tal fine abbiamo ottimizzato i protocolli per la loro purificazione mediante citometria di flusso dal muscolo dissociato enzimaticamente, che illustrerà in questo articolo. Le popolazioni ottenuti usando questo metodo contenere cellule colonia miogeniche o fibro / adipogenico formando: non altri tipi di cellule sono in grado di espandere in vitro o sopravvivere in consegna vivo. Tuttavia, quando queste popolazioni vengono utilizzati subito dopo l&#39;ordinamento per l&#39;analisi molecolare (ad es qRT-PCR) si deve tenere presente che i sottoinsiemi appena ordinati può contenere altre cellule agente inquinante che non hanno la capacità di formare colonie o engrafting destinatari.

Watch this Scientific Journal Video about Purification of Progenitors from Skeletal Muscle at JoVE.com

Purification of Progenitors from Skeletal Muscle

Metodo per la dissociazione enzimatica, l&#39;etichettatura e la purificazione della superficie mediante citometria a flusso di fibro / adipogenico e progenitori miogenici da topo muscolo scheletrico.

Purificazione di progenitori dal muscolo scheletrico

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

purification-of-progenitors-from-skeletal-muscle

Research

JoVE Journal

Biology

18.2K Views.  University of British Columbia. Metodo per la dissociazione enzimatica, l'etichettatura e la purificazione della superficie mediante citometria a flusso di fibro / adipogenico e progenitori miogenici da topo muscolo scheletrico.

Video: Purification of Progenitors 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

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

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 ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Basato sulla fluorescenza Misura Store-Entry operato di calcio in cellule vive: dal Cancer Cell coltura per fibra muscolare scheletrica

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-lesioni del muscolo scheletrico embrionale Zebrafish

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

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 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 ...

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

Identificazione, isolamento e caratterizzazione di progenitori fibro-adipogeni (FAPs) e progenitori miogeni (MP) nel muscolo scheletrico nel ratto

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

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 ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.