Name: isolation and characterization of exosomes from skeletal muscle fibroblasts
Uploaded: 2020-05-16T13:30:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Alessandra d'Azzo har Jewelers for Children (JFC) Endowed Chair i genetik og genterapi. Dette arbejde blev delvist støttet af NIH-tilskud R01GM104981, RO1DK095169 og CA021764, Assisi Foundation of Memphis og American Libanese Syrian Associated Charities.

Alle procedurer i mus blev udført i henhold til dyreprotokoller godkendt af St. Jude Children's Research Hospital, Institutional Animal Care and Use Committee og National Institutes of Health retningslinjer.
1. Fremstilling af opløsninger og medier
<ol><li>Forbered fordøjelsesopløsningen ved at blande 15,4 ml PBS med 2,5 ml 20 mg/ml collagenase P (5 mg/ml slutkoncentration), 2 ml 11 E/ml dispase II (1,2 E/ml slutkoncentration) og 100 μL 1,0 MCaCl2 (5 mM slutkoncentration).</l...

<ol>
	<li>Doyle, L. M., Wang, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+Extracellular+Vesicles,+Their+Origin,+Composition,+Purpose,+and+Methods+for+Exosome+Isolation+and+Analysis.">Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis.</a> Cells. 8 (7), 727 (2019).</li><li>Gurunanthan, S., Kang, M. H., Jeyaraj, M., Qasim, M., Kim, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+Isolation,+Characterization,+Biological+Function,+and+Multifarious+Therapeutic+Approaches+of+Exosomes.">Review of the Isolation, Characterization, Biological Function, and Multifarious Therapeutic Approaches of Exosomes.</a> Cells. 8 (4), 307 (2019).</li><li>Zhang, Y., Liu, Y., Liu, H., Tang, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+biogenesis,+biologic+function+and+clinical+potential.">Exosomes: biogenesis, biologic function and clinical potential.</a> Cell &amp; Bioscience. 9 (19), 3070-3085 (2019).</li><li>van de Vlekkert, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+exosome+release+is+the+pathogenic+pathway+linking+a+lysosomal+deficiency+to+generalized+fibrosis.">Excessive exosome release is the pathogenic pathway linking a lysosomal deficiency to generalized fibrosis.</a> Science Advances. 5 (7), 3270 (2019).</li><li>Rackov, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Vesicle-Mediated+Control+of+Cell+Function:+The+Role+of+Extracellular+Matrix+and+Microenvironment.">A Vesicle-Mediated Control of Cell Function: The Role of Extracellular Matrix and Microenvironment.</a> Frontiers in Physiology. 9, 651 (2018).</li><li>Howitt, J., Hill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+the+pathology+of+neurodegenerative+diseases.">Exosomes in the pathology of neurodegenerative diseases.</a> Journal of Biological Chemistry. 291 (52), 26589-26597 (2016).</li><li>Jing, H., Tang, S., Lin, S., Liao, M., Chen, H., Zhou, J. <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+extracellular+vesicles+in+renal+fibrosis.">The role of extracellular vesicles in renal fibrosis.</a> Cell Death and Disease. 10 (5), 367 (2019).</li><li>Asef, A., Mortaz, E., Jamaati, H., Velayati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+role+of+extracellular+vesicles+and+exosomes+in+the+pathogenesis+of+cystic+fibrosis.">Immunologic role of extracellular vesicles and exosomes in the pathogenesis of cystic fibrosis.</a> Journal of Respiratory Diseases, Thoracic Surgery, Intensive Care and Tuberculosis. 17 (2), 66-72 (2018).</li><li>Cai, A., Cheng, X., Pan, X., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+exosomes+in+liver+physiology+and+pathology.">Emerging role of exosomes in liver physiology and pathology.</a> Hepatology Research. 47 (2), 194-203 (2017).</li><li>Machado, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+lysosomal+exocytosis+mediates+cancer+progression.">Regulated lysosomal exocytosis mediates cancer progression.</a> Science Advances. 1 (11), 1500603 (2015).</li><li>Tian, W., Liu, S., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+role+of+exosomes+in+cancer+metastasis.">Potential role of exosomes in cancer metastasis.</a> Biomed Research International. , 4649705 (2019).</li><li>Barile, L., Vassalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+Therapy+delivery+tools+and+biomarkers+of+disease.">Exosomes: Therapy delivery tools and biomarkers of disease.</a> Pharmacology and Therapeutics. 174, 63-78 (2017).</li><li>Patel, G. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+exosome+isolation+methods+using+culture+supernatant+for+optimum+yield,+purity+and+downstream+applications.">Comparative analysis of exosome isolation methods using culture supernatant for optimum yield, purity and downstream applications.</a> Science Reports. 9 (1), 5335 (2019).</li><li>Ayala-Mar, S., Donoso-Quezada, J., Gallo-Villanueva, R. C., Perez-Gonzalez, V. H., Gonz&#225;lez-Valdez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+and+challenges+in+the+recovery+and+purification+of+cellular+exosomes.">Recent advances and challenges in the recovery and purification of cellular exosomes.</a> Electrophoresis. 40 (23-24), 3036-3049 (2019).</li><li>Boutagy, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Mitochondria+from+Minimal+Quantities+of+Mouse+Skeletal+Muscle+for+High+Throughput+Microplate+Respiratory+Measurements.">Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements.</a> Journal of Visualized Experiments. (105), e53217 (2015).</li><li>. Muscle Dissection in mouse Available from: <a target="_blank" href="https://www.youtube.com/watch?v=Wh0gXfrHaH8">https://www.youtube.com/watch?v=Wh0gXfrHaH8</a> (2016)</li><li>Mas-Bargues, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+oxygen+concentration+in+stem+cell+culture+for+regenerative+medicine.">Relevance of oxygen concentration in stem cell culture for regenerative medicine.</a> International Journal of Molecular Sciences. 20 (5), 1195 (2019).</li><li>Bois, P. R., Grosveld, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FKHR+(FOX01a)+is+required+for+myotube+fusion+of+primary+mouse+myoblasts.">FKHR (FOX01a) is required for myotube fusion of primary mouse myoblasts.</a> The EMBO Journal. 22 (5), 1147-1157 (2003).</li><li>Machida, S., Spangenburg, E. E., Booth, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+rat+muscle+progenitor+cells+have+decreased+proliferation+and+myotube+formation+during+passages.">Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages.</a> Cell Proliferation. 37, 267-277 (2004).</li><li>Syverud, B. C., Lee, J. D., VanDusen, K. W., Larkin, L. M. <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+purification+of+satellite+cells+for+skeletal+muscle+tissue+engineering.">Isolation and purification of satellite cells for skeletal muscle tissue engineering.</a> Journal of Regenerative Medicine. 3 (2), 117 (2014).</li></ol>

Et kritisk skridt for en vellykket isolering af exosomer fra kulturmedierne, som skitseret i denne protokol, er korrekt etablering og vedligeholdelse af primære musefibroblastkulturer fra voksne skeletmuskler. Disse kulturer skal opretholdes på et lavt iltniveau for at sikre fysiologisk-lignende forhold (O2-niveau i skeletmuskulatur er ~ 2,5%)15. Primære fibroblaster vil ændre egenskaber, når de overføres i kultur for mange gange. Derfor er et lavt passagetal afgørende for ideelt...

Exosomer er heterogene ekstracellulære vesikler, der varierer i størrelse fra 30-150 nm. De er etablerede nøgleaktører i fysiologiske og patologiske processer på grund af deres allestedsnærværende fordeling i væv og organer 1,2. Exosomer bærer en kompleks last af proteiner, lipider, DNA-typer og RNA-typer, som varierer alt efter den type celler, hvorfra de stammer 1,2,3. Exosomer er beriget med proteiner, der har forskellige funktioner (dvs. tetraspaniner, herunder CD9 og CD63) er ansvarlige for fusionsbegivenheder. For eksempel er varmechokproteiner HSP70 og HSP90 involveret i antigenbinding og præsentation. Derudover deltager Alix, Tsg101 og flotillin i exosombiogenese og frigivelse og bruges i vid udstrækning som markører for disse nanovesikler 2,3,4.
Exosomer indeholder også en række RNA'er (dvs. mikroRNA'er, lange ikke-kodende RNA'er, ribosomale RNA'er), der kan overføres til modtagerceller, hvor de påvirker nedstrøms signalering3. At være omgivet af en enkelt enhedsmembran afhænger exosombioaktivitet ikke kun af lasten af proteiner og nukleinsyrer, men også på lipidkomponenter i den begrænsende membran1. Exosomale membraner er beriget med phosphatidylserin, phosphatidsyre, kolesterol, sphingomyelin, arachidonsyre og andre fedtsyrer, som alle kan påvirke exosomstabilitet og membrantopologi 2,3. Som et resultat af last- og lipidarrangementet initierer eksosomer signalveje i modtagende celler og deltager i vedligeholdelsen af normal vævsfysiologi 1,2,4,5. Under visse patologiske tilstande (dvs. neurodegeneration, fibrose og kræft) har de vist sig at udløse og udbrede patologiske stimuli 4,6,7,8,9,10,11.
På grund af deres evne til at sprede signaler til nærliggende eller fjerne steder er exosomer blevet værdifulde biomarkører til diagnosticering eller prognose af sygdomstilstande. Derudover er exosomer blevet anvendt eksperimentelt som bærere af terapeutiske forbindelser 2,12. Den potentielle anvendelse af disse nanovesikler i klinikken gør isolationsmetoden stadig vigtigere for at opnå maksimalt udbytte, renhed og reproducerbarhed. Forskellige teknikker til isolering af exosomer er blevet udviklet og implementeret. Generelt kan exosomer isoleres fra konditionerede cellekulturmedier eller kropsvæsker ved differentiel centrifugering, størrelsesekskluderingskromatografi og immunindfangning (ved hjælp af kommercielt tilgængelige kits). Hver tilgang har unikke fordele og ulemper, der tidligere er blevet diskuteret 1,2,13,14.
Den skitserede protokol fokuserer på 1) isolering og dyrkning af primære fibroblaster fra voksne mus gastrocnemius muskel og 2) oprensning og karakterisering af exosomer frigivet i kulturmediet af disse celler. En veletableret protokol til isolering af exosomer fra primære fibroblaster til funktionelle undersøgelser mangler i øjeblikket. Primære fibroblaster udskiller ikke store mængder exosomer, hvilket gør isolerings- og rensningsprocessen udfordrende. Denne protokol beskriver oprensningen af store mængder rene exosomer fra store kulturvolumener, samtidig med at deres morfologiske integritet og funktionelle aktivitet opretholdes. Oprensede exosomer opnået fra konditioneret medium er blevet anvendt med succes i in vitro-optagelsesforsøg for at inducere specifikke signalveje i modtagerceller. De er også blevet brugt til komparative proteomiske analyser af exosomale laster fra flere biologiske prøver4.

<table>
	<tbody>
		<tr>
			<td>10 cm dishes</td>
			<td>Midwest Scientific, TPP</td>
			<td>TP93100</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm dishes</td>
			<td>Midwest Scientific</td>
			<td>TP93150</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific, Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin Fraction V</td>
			<td>Roche</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5430R with rotors FA-35-6-30/ FA-45-48-11</td>
			<td>Eppendorf</td>
			<td>022620659/5427754008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemidoc MP imaging system</td>
			<td>BioRad</td>
			<td>12003154</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase P</td>
			<td>Sigma, Roche</td>
			<td>11 213 857 001</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>cOmplete protease inhibitor cocktail</td>
			<td>Millipore/Sigma, Roche</td>
			<td>11697498001</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion Blotter with plate electrodes</td>
			<td>BioRad</td>
			<td>1704070</td>
			<td></td>
		</tr>
		<tr>
			<td>Criterion TGX stain-free protein gel</td>
			<td>BioRad</td>
			<td>5678034</td>
			<td>10% 18-well, midi-gel</td>
		</tr>
		<tr>
			<td>Criterion vertical electrophoresis cell (midi)</td>
			<td>BioRad</td>
			<td>NC0165100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma, Roche</td>
			<td>04 942 078 001</td>
			<td>neutral protease, grade II</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma/Millipore, Roche</td>
			<td>10708984001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modification Eagles Medium</td>
			<td>Corning</td>
			<td>15-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Pharmco by Greenfield Global</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical centrifuge tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluostar Omega multi-mode microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX supplement</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Immobilon-P Transfer membranes</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli sample buffer (4x)</td>
			<td>BioRad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate solution</td>
			<td>Sigma</td>
			<td>63052-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>LabScientific</td>
			<td>M-0842</td>
			<td></td>
		</tr>
		<tr>
			<td>O2/CO2 incubator</td>
			<td>Sanyo</td>
			<td>MC0-18M</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Premium microcentrifuge tubes</td>
			<td>Fisher Scientific, Midwest Scientific</td>
			<td>AVSC1510</td>
			<td>1.7 mL</td>
		</tr>
		<tr>
			<td>Protected disposable scalpels</td>
			<td>Fisher Scientific, Aspen Surgical Bard-Parker</td>
			<td>372610</td>
			<td></td>
		</tr>
		<tr>
			<td>Running buffer</td>
			<td>BioRad</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick release-GP sterile vacuum filtration system</td>
			<td>Millipore</td>
			<td>S2GPU05RE</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Sterile cell strainer (70 mm)</td>
			<td>Fisher Scientific, Fisher brand</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific, Fisher Chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal west Femto</td>
			<td>Thermo Fisher Scientific</td>
			<td>34096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin wall Polypropylene tubes</td>
			<td>Beckman Coulter</td>
			<td>326823</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer buffer</td>
			<td>BioRad</td>
			<td>16110734</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma</td>
			<td>91228-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>BioRad</td>
			<td>1610719</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100 solution</td>
			<td>Sigma</td>
			<td>93443-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Thermo Fisher Scientific, Gibco</td>
			<td>12604-013</td>
			<td>No phenol red</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>BioRad</td>
			<td>#1610781</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge Optima XPM</td>
			<td>Beckman Coulter</td>
			<td>A99842</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tube (14x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-clear tubes (25x89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath Isotemp 220</td>
			<td>Fisher Scientific</td>
			<td>FS220</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of exosomes from skeletal muscle fibroblasts

Exosomer er små ekstracellulære vesikler frigivet af stort set alle celler og udskilles i alle biologiske væsker. Mange metoder er blevet udviklet til isolering af disse vesikler, herunder ultracentrifugering, ultrafiltrering og størrelseseksklusionskromatografi. Imidlertid er ikke alle egnede til storskala eksosomrensning og karakterisering. Skitseret her er en protokol til etablering af kulturer af primære fibroblaster isoleret fra voksne museskeletmuskler, efterfulgt af oprensning og karakterisering af exosomer fra disse cellers kulturmedier. Metoden er baseret på anvendelse af sekventielle centrifugeringstrin efterfulgt af saccharosedensitetsgradienter. Renheden af de exosomale præparater valideres derefter ved western blot-analyser ved hjælp af et batteri af kanoniske markører (dvs. Alix, CD9 og CD81). Protokollen beskriver, hvordan man isolerer og koncentrerer bioaktive exosomer til elektronmikroskopi, massespektrometri og optagelsesforsøg til funktionelle studier. Det kan let skaleres op eller ned og tilpasses til exosomisolering fra forskellige celletyper, væv og biologiske væsker.

Denne protokol er egnet til rensning af exosomer fra store mængder konditioneret medium på en omkostningseffektiv måde. Proceduren er meget reproducerbar og konsekvent. Figur 1 viser transmissionselektronmikroskopi (TEM) billede af exosomer renset fra dyrkningsmediet af musens primære fibroblaster. Figur 2 viser proteinekspressionsmønsteret for kanoniske exosomale markører og fraværet af cytosoliske (LDH) og ER (calnexin) proteinforurenende stoffer. <stro...

Watch this Scientific Journal Video about Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts at JoVE.com

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Denne protokol illustrerer 1) isolering og dyrkning af primære fibroblaster fra den voksne muses gastrocnemiusmuskel samt 2) oprensning og karakterisering af exosomer ved hjælp af en differentiel ultracentrifugeringsmetode kombineret med saccharosedensitetsgradienter efterfulgt af western blot-analyser.

Isolering og karakterisering af exosomer fra skeletmuskelfibroblaster

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

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Biology

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 ...

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 ...

Purification of Progenitors from Skeletal Muscle

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

Isolation and Characterization of RNA-Containing Exosomes

Isolering og karakterisering af RNA-Varer Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

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

Isolering og dyrkning af individuelle myofibre og deres satellit celler fra voksne skeletmuskler

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

Isolering af blod-fartøj-afledt Multipotente Forstadier fra human skeletmuskel

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

Isolering af mitokondrier fra minimale mængder af Mouse Skeletmuskel for High Throughput Microplate Respiratory Målinger

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

Isolering af Intakt Mitokondrier fra Skeletal Muscle ved differentialcentrifugering for høj opløsning respirometri Målinger

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 Adult Cardiac Fibroblasts and Myofibroblasts

Isolation og karakterisering af voksne hjertefibroblaster og Myofibroblaster

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

Isolering af humane ventrikulære kardiomyocytter fra 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 ...