1. Collection de tissus: <ol><li> Utilisez la souris entre 7 et 12 semaines. Habituellement, au moins deux animaux sont utilisés: l&#39;un est de mettre en place le FACS seule couleur contrôles requis pour l&#39;indemnisation et pour la fluorescence-moins-un échantillons de contrôle isotypique. L&#39;autre est l&#39;échantillon réel pour le tri. </li><li> Euthanasier les souris par inhalation de CO 2 ou selon un protocole d&#39;éthique des choix. Placez la souris sur une serviette en papier, face vers le haut, et de pulvérisation avec de l&#39;éthanol à 70% à fond pour éviter toute contamination avec la fourrure de la souris. </li><li> Soulever la peau dans la région abdominale avec une pince, puis prendre une petite coupe longitudinale avec des ciseaux pointus. </li><li> Peler les deux volets de la peau dans des directions opposées (haut et bas) complètement exposer les muscles des membres postérieurs en dessous. </li><li> D&#39;accise du tissu musculaire en insérant et en étendant les ciseaux le long des deux côtés du tibia. </li><li> Répétez le même processus pour le fémur pour en extraire le tissu musculaire. </li><li> Placez tous les tissus musculaires excisées dans 60mm Pétri-vaisselle, en utilisant un seul plat pour le tissu de chaque souris. </li><li> Répéter l&#39;opération pour toutes les souris. </li></ol> 2. Dissocier tissu en cellules isolées par digestion enzymatique: Tout au long de cette section du protocole, l&#39;utilisation des réactifs stériles et des outils et du travail dans des conditions stériles. <ol start="9"><li> Ajouter 2 ml de collagénase II (Sigma, cat # 6885, 500U / ml) et 20 ul de CaCl 2 250mm d&#39;actions à chaque boîte de Petri. </li><li> Déchirer le tissu musculaire en petits morceaux (environ 1 mm 3) avec deux pinces (une dentelé, une avec 2 dents, des outils de Fine Science, cat # 11051-10, 11154-10), en prêtant attention à enlever et jeter autant contaminés non tissu musculaire que possible. </li><li> Couvrir le Petri-vaisselle et les incuber à 37 degrés Celsius pendant 30 minutes. </li><li> Récupérer le Petri-plats et d&#39;utiliser une seringue de 3 ml plongeur au fond écraser les morceaux de tissu. Utilisez un poussoir pour chaque échantillon afin d&#39;éviter les contaminations. </li><li> Ajoutez un peu (~ 5 ml) stériles PBS froid à chaque boîte de Petri et le transfert des boues de tissu dans un tube de 50 ml Falcon (rincer la boîte de Petri, si nécessaires pour récupérer tous les tissus) </li><li> Agiter le tube Falcon vigoureusement, remplir le tube avec du PBS, centrifuger @ 800rpm x 5 minutes (Modèle de paillasse Eppendorf: 5810R), et aspirer le surnageant (répéter 3 fois) </li><li> Ajouter le mélange de 1 ml de collagénase D (Roche, Cat # 11088882001, 1,5 U / ml) / dispase II (Roche, Cat #: 04942078001, 2.4u / ml) et 10 ul de CaCl 2 (250mm) à chaque tube, incuber à 37 degrés Celsius avec rotation pendant une heure. </li><li> Ajouter 5-10 mL de froid, de PBS stérile, vortex et une pipette soigneusement afin d&#39;homogénéiser le tissu. </li><li> Remplir les tubes avec du PBS et mélangez bien. </li><li> Transférer le liquide dans un tamis cellulaire 40μm placé sur le dessus d&#39;un tube Falcon de 50 ml de filtrer les morceaux restants grande partie des tissus. Diviser chaque échantillon homogène dans des tubes Falcon 50 ml trois. Remplissez chacun des tubes avec du PBS, puis centrifuger @ 1600RPM pendant 5 minutes à 8 degrés Celsius. </li><li> Jeter le surnageant et de recueillir des cellules pour la coloration. </li></ol> 3. Coloration des anticorps et de tri: Tout au long de cette section du protocole, l&#39;utilisation FACS stérile et tampons de collecte et de travailler dans des conditions stériles. Anticorps commerciaux sont généralement considérés comme stériles si elles sont correctement traitées. <ol start="20"><li> Pour seule couleur et d&#39;échantillons de contrôle isotype, resuspendre l&#39;échantillon dans un tampon de FACS 1ml et diviser les cellules en suspension dans neuf 1.5ml tubes Eppendorf pré-étiquetées (100 ul de chaque tube). <ol><li> Mettre en place des taches de couleurs simples mélanges selon le tableau suivant: ATTENTION: La concentration finale d&#39;anticorps isotype contrôle de la tache doit correspondre à celle de l&#39;anticorps primaire, ils sont le contrôle des. Par exemple, si l&#39;anti-Sca-1 est utilisé pour une μg/10 6 cellules, son isotype correspondant doit être utilisé à la même concentration. <table border="1"><tbody><tr><td> Tube # </td><td> Nom du tube </td><td> Société </td><td> Cat # </td><td> clone </td><td> isotype </td><td> de dilution </td></tr><tr><td> 1 </td><td> Non-taches </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> Maison faite </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> Maison faite </td><td></td><td> R2F2 </td><td> Rat IgG2b </td><td> 1000 </td></tr></tbody></table></li><li> Mettre en place coloration contrôle isotypique mixages en utilisant le tableau suivant: <table border="1"><tbody><tr><td> Tube # </td><td> Nom du tube </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> Déposer le mélange de couleurs simples et isotopiques coloration de contrôle (nous avons l&#39;habitude d&#39;ajuster la concentration d&#39;anticorps de sorte qu&#39;entre 5 et 10 uL de coloration mélange sont ajoutés à chaque échantillon) dans le tube Eppendorf appropriée de l&#39;étape 20. Bien mélanger et incuber sur la glace pendant 1 heure. </li></ol></li><li> Colorer des cellules de souris échantillon avec 800μL du cocktail d&#39;anticorps (ci-dessous) pour le tri de cellules FACS. Bien mélanger et incuber sur la glace pendant 1 heure. Cocktail d&#39;anticorps: Hoechst, PI, CD31-FITC, CD45-FITC, SCA1-PECY7, α-7 APC ATTENTION: Le rapport optimal des anticorps aux cellules doit être déterminée par titration individuelle chaque anticorps, comme des différences significatives peuvent exister entre les lots. La quantité optimale d&#39;anticorps à utiliser doit être exprimée en μgs d&#39;anticorps par 10 6 cellules cibles et maintenue constante lors de l&#39;utilisation du même lot d&#39;anticorps. En clair, si le nombre de cellules cibles ne change pas significativement entre les expériences, ou si le volume est réduit avec coloration du nombre de cellules, une dilution fixe de chaque anticorps peut également être utilisé. </li><li> Lavage: Ajouter environ 1 ml de tampon FACS à chacun des 9 tubes de contrôle; ajouter environ 20 ml de tampon FACS au tube échantillon. </li><li> Centrifuger les tubes Eppendorf @ 3000rpm pendant 5 minutes (banc Eppendorf centrifugeuse, Modèle: 5417C). Centrifuger le tube de l&#39;échantillon @ 1600RPM pendant 5 minutes (banc Eppendorf dessus centrifugeuse, Modèle: 5810R). </li><li> Aspirer et jeter le surnageant de tous les tubes. </li><li> Resuspendre les cellules dans le tampon FACS (1ml pour chacun des contrôles, 4ml pour le tri des échantillons), les cellules Pipeter dans des «tubes FACS&quot; à travers le tamis cellulaire de la PAC (cat # 352235 BD Falcon, 40μm taille des pores) pour enlever les grumeaux restants qui peuvent affecter le cytomètre. </li><li> Mettre en place la trieuse FACS avec des contrôles seule couleur et de contrôle isotypique. </li><li> Recueillir les cellules triées selon le tableau suivant dans un tampon 3,5 ml de collecte (95% DMEM, 5% de FBS) séparément. Normalement, lorsque la récolte deux membres postérieurs, une souris de type sauvage unique pourrait produire environ 150.000 cellules progénitrices myogéniques ou 100.000 cellules progénitrices adipogéniques, avec la viabilité supérieure à 95%, tel que jugé par réanalyse cellules triées par FACS à la fin de la procédure (figure 1 ) Définition des progéniteurs myogéniques et adipogéniques basée sur coloration de surface <table border="1"><tbody><tr><td> Anticorps / teinture </td><td> Progénitrices myogéniques (MP) </td><td> Progénitrices adipogéniques (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. Transplantation: <ol start="28"><li> Centrifuger les cellules triées @ 1500rpm pendant 5 minutes, retirer les cellules surnageant et transfert à l&#39;autoclave des tubes Eppendorf (1,5 ml), laver les cellules avec 500 ul de PBS stérile (sérum gratuit!), Puis centrifuger @ 3000rpm pendant 5 minutes. Maintenir tous les réactifs dans des conditions stériles et de travailler dans une hotte de culture tissulaire, resuspendre progéniteurs myogéniques dans le PBS, ou progénitrices adipogéniques de Matrigel (cat # 354234 BD) à environ 10 6 cellules / ml. </li><li> Transplant progéniteurs myogéniques ou progénitrices adipogéniques aux souris receveuses. <ol><li> Anesthésier la souris avec isoflouran en fonction de votre politique de l&#39;institution. </li><li> Raser les poils autour de la région d&#39;injection, puis stériliser la peau par frottement avec de l&#39;éthanol à 70% </li><li> Injecter 20 pi des progéniteurs myogéniques ou progéniteurs adipogéniques (= 20k cellules) à l&#39;aide d&#39;une seringue 3 / 10 CC insuline (BD # 309300 cat). </li></ol></li><li> Après un moment approprié (trois semaines qui fonctionne bien, mais myofibrilles exprimer provenant de donneurs marqueurs transgéniques sont habituellement évidents dans la semaine après la transplantation), d&#39;anesthésier les souris receveuses de l&#39;étape 29 par injection intrapéritonéale de 400 ul Avertin (Cat Sigma # T04840-2, 25 mg / ml), puis perfuser transcardiaque avec 50mL de PBS (contient 10 pM de l&#39;EDTA) en premier, suivis de 15 ml de 4% en PFA. Recueillir tissu cible, le post-fixer avec 4% PFA nuit si nécessaire, puis transfert à 20% de sucrose pendant la nuit. Intégrer dans les PTOM, geler et cryosection. </li></ol> 5. Les résultats représentatifs: Lorsque MP sont transplantés dans les muscles squelettiques de souris, ils facilement fusionner avec pré-existante myofibrilles. Par conséquent, toute étiquette génétique présente dans les cellules du donneur sera facilement détectable dans les fibres qui les a reçus. La figure 2 montre un exemple où les cellules transplantées ont exprimé humaine phostphatase alcalines, a révélé histochimiquement. Lorsque l&#39;AP sont transplantés le résultat est très dépendante de l&#39;environnement du site de transplantation: lorsqu&#39;elles sont transplantées sous-cutanée de ces cellules donnent origines en adipocytes et les myofibroblastes (voir figure 2). Dans de nombreux autres sites, y compris les muscles, ils ne survivent pas à moins dégénérescence graisseuse a été induite 3. <img src="/files/ftp_upload/2476/2476fig1.jpg" alt="Figure 1" /> Figure 1. Tri stratégie pour l&#39;isolement des populations progénitrices du muscle squelettique stratégie de tri (A):. Cellules viables ont été identifiés sur la base de dispersion vers l&#39;avant et diffusion latérale. Coloration Hoechst a été utilisé pour exclure les débris anucléées et de l&#39;iodure de propidium (PI) coloration d&#39;exclure les cellules mortes. Hématopoïétiques (CD45) et endothéliales (CD31) des cellules ont été exclus de la porte de tri. Le α7 + Hoechst + PI - CD45 - CD31 - Scal - sous-ensemble contient tous les progéniteurs myogéniques (MP). Le Scal + Hoechst + PI - CD45 - CD31 - α7 - population contient des progéniteurs adipogéniques (AP). (B) Fluorescence-moins-un contrôle isotype (FMO) confirment la spécificité de la coloration. (C) Pureté contrôles de sous-ensembles MP et AP après le tri. <img src="/files/ftp_upload/2476/2476fig2.jpg" alt="Figure 2" /> Figure 2. Les résultats représentatifs suivants MP et AP transplantation. AP ont été transplantés sous-cutanée (panneau supérieur) et le député par voie intramusculaire (panneau inférieur). Dans les deux cas, les cellules du donneur provenaient d&#39;une souris transgéniques exprimant la phosphatase alcaline humaine, identifié par la coloration brune.

<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>

L&#39;objectif de ce protocole est de trouver un équilibre raisonnable entre les rendements élevés et une viabilité élevée des cellules purifiées. L&#39;étape la plus critique pour s&#39;assurer que les cellules saines sont récupérés est, sans surprise, la dissociation enzymatique du tissu de départ. La manipulation des tissus doivent être particulièrement doux, ce qui est difficile à démontrer ou à apprécier encore dans un format audiovisuel. Un autre facteur clé est la longueur de la procédure. Plus il faut aller chez le donneur à l&#39;animal receveur, le bas de la viabilité et donc l&#39;efficacité greffe. Faut-il y avoir aucune question sur la viabilité qui n&#39;est pas immédiatement répondu en regardant la fréquence des PI événement positif dans les échantillons de cellules purifiées après le tri, nous suggérons qu&#39;un test de dilution limite à mesurer clonogénicité est effectué 3. Sortes typiques du muscle intact régulièrement le rendement d&#39;environ 1 sur 15 à 20 cellules capables d&#39;initier des colonies myogénique ou fibro / adipogéniques in vitro. Alors que pratiquement 100% des colonies initiée par les députés contiennent les myotubes différenciés, seulement environ un tiers des colonies obtenues à partir des adipocytes FAP contiennent, en plus de douceur myofibroblastes actine musculaire positif.

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Collagénase II </td><td> Sigma-Aldrich </td><td> C-6885, </td><td> 500 U / ml </td></tr><tr><td> Collagénase D </td><td> Roche </td><td> 11088882001 </td><td> 1,5 U / ml </td></tr><tr><td> Déplacer II </td><td> Roche </td><td> 04942078001 </td><td> 2,4 U / ml </td></tr><tr><td> tamis cellulaire </td><td> BD </td><td> 352340 </td><td> 40 um </td></tr><tr><td> Tube avec filtre cellulaire </td><td> BD </td><td> 352235 </td><td> 40 um </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> Fait maison </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 intégrine APC </td><td> Fait maison </td><td> Renseignez-vous auprès du laboratoire Rossi </td><td> R2F2 </td></tr></tbody></table>

purification of progenitors from skeletal muscle

Le muscle squelettique contient populations progénitrices multiples origines distinctes embryonnaire et potentiel de développement. Progéniteurs myogéniques, résidant habituellement dans une «position de cellule satellite» entre la membrane plasmatique fibres musculaires et de la membrane basale de laminine-riche qu&#39;il ensheaths, sont auto-renouvellement des cellules qui sont uniquement engagés à 1,2 lignage musculaire. Nous avons récemment décrit une deuxième classe de navire de progéniteurs associés qui peuvent générer des myofibroblastes et les adipocytes blancs, qui répond efficacement aux dommages causés par la prolifération d&#39;entrer et offre un soutien trophique à cellules myogéniques pendant 3,4 régénération des tissus. L&#39;un des tests les plus fiables pour déterminer le potentiel de développement et de régénération d&#39;une population cellulaire donnée repose sur leur isolement et de la transplantation 5-7. À cette fin, nous avons optimisé les protocoles pour leur purification par cytométrie en flux à partir de muscle enzymatiquement dissocié, ce qui nous donnera un aperçu dans cet article. Les populations obtenues en utilisant cette méthode, soit des cellules contiennent des colonies myogénique ou fibro / adipogéniques formant: pas d&#39;autres types cellulaires sont capables de développer in vitro ou in vivo dans la prestation de survivant. Toutefois, lorsque ces populations sont utilisées immédiatement après le genre d&#39;analyse moléculaire (par exemple qRT-PCR), il faut garder à l&#39;esprit que les sous-ensembles fraîchement triées peut contenir d&#39;autres cellules contaminant qui n&#39;ont pas la capacité de former des colonies ou greffant les bénéficiaires.

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

Purification of Progenitors from Skeletal Muscle

Méthode pour la dissociation enzymatique, l&#39;étiquetage de surface et de la purification par cytométrie en flux de fibro / adipogéniques et les progéniteurs myogéniques du muscle squelettique murin.

Purification des progéniteurs du muscle squelettique

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

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18.2K vues.  University of British Columbia. Méthode pour la dissociation enzymatique, l'étiquetage de surface et de la purification par cytométrie en flux de fibro / adipogéniques et les progéniteurs myogéniques du muscle squelettique murin.

Vidéo: 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.

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

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.

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 ...

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.

Basées sur la fluorescence de mesure de l&#39;entrée du calcium magasin fonctionnant dans des cellules vivantes: à partir de cellules cancéreuses en culture de fibre musculaire squelettique

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-infligé des blessures de poisson zèbre muscle squelettique embryonnaire

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.

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 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 ...

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.

Identification, isolement et caractérisation des progéniteurs fibro-adipogéniques (PAF) et des progéniteurs myogéniques (MP) dans le muscle squelettique chez le rat

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.

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 ...

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.