Yazarlar akış sitometri ile onu mükemmel teknik yardım için Alison Logar teşekkür etmek istiyorum. Bu çalışma Savunma Bakanlığı (JH), Henry J. Mankin Endowed Başkanı (JH), Eğitim ve Kazakistan Cumhuriyeti Bilim Bakanlığı (AS) hibe ile desteklenmiştir. CWC Amerikan Kalp Derneği predoctoral bursu (11PRE7490001) tarafından kısmen desteklenmiştir. M.Corselli Rejeneratif Tıp eğitim bursu için California Institute (TG2-01169) tarafından desteklenmiştir.

1. Kas Biyopsi İşleme <ol><li> Dulbecco Modifiye Edilmiş Eagle Ortamı,% 5 fetal sığır serumu (FBS) ile desteklenmiş (DMEM) içinde buz üzerinde insan iskelet kası biyopsisi Koruma% 1 Penisilin-Streptomisin taşıma sırasında (P / S). </li><li> Kas biyopsi alınmasından sonra, taşıma kabından örnek çıkarın ve steril şartlar altında% 2 antifungal antibiyotik solüsyon (A / A) ile doldurulmuş fosfat-tamponlu salin (PBS) içinde iki kere yıkayın. </li><li> 2% A / A ile takviye edilmiş DMEM içinde steril makas ve forsepsle bağlı yağ ve bağ doku çıkarın. </li><li> Diseksiyon mikroskobu altında büyük kan damarları sökülmesi ve daha sonra küçük parçalar (boyut &lt;1 cm2) içine, kas numune kesin. </li><li> 20 mi muhafaza ortamında kesme kas parça (&lt;5 gram) korumak (PM, DMEM,% 10 FBS ve% 1 P / S ile takviye edilmiş) kadar 5 gün boyunca 4 ℃ de. </li></ol> 2. Kas Dissociatiyonu ve Hücre İzolasyonu <ol><li> Hücre izolasyon gününde, PM kas parçaları kaldırmak ve PBS içinde iki kez yıkayın% 2 P / S ile desteklenmiş. Sindirim solüsyonu yapmak için, tip I, tip II ve tip IV kolajenazları taze PM halinde (100 mg / ml) ilave edilmektedir. </li><li> Ince ince doğrayın ve çözüm hiçbir pıhtılaşması ile 10 ml serolojik pipet geçene kadar mekanik PM küçük bir miktar ile bir petri kabına steril makas ve forseps ile kas parçaları kıyma. Bu işlem sırasında kalan yağ ve bağ doku çıkarın. Yetişkin kas en az 8-10 gram ya da her bir hücre izolasyonu fetal kas 2 gram kullanın. </li><li> 20 ml (veya 10 ml cenin kıyılmış kas 2 gram) 10 ml&#39;lik bir pipet ile serolojik sindirim solüsyonunun kesitler kıyılır yetişkin kas 4-5 gram aktarın. 80 rpm - 70 ° C&#39;de bir orbital çalkalayıcı üzerinde 37 ° C&#39;de 60 dakika ℃ - 50 Digest. Buna göre miktarına bağlı olarak sindirim süresi ve / veya çalkalama hızının ayarlanmasıyla, hücre yüzey antijeni, verim ve koruma optimizedoku. Bulanıklık neredeyse silinene kadar sindirim durumunu her 15-20 dk gözlemleyin. </li><li> Şiddetle sindirilmiş dokuya 10 ml serolojik pipet ile 3-5 kez pipetle. Reaksiyonu durdurmak ve daha sonra 4 dakika boyunca 400 x g&#39;de santrifüj PM eşit miktarda ekleyin. </li><li> Dikkatlice süpernatant kaldırmak; yıkamak için 10 mL PM olarak pelet, ve daha sonra 100 um&#39;lik bir hücre süzgecinden süzülmüştür. </li><li> 4 dakika boyunca 400 x g&#39;de santrifüj ve dikkatle süpernatant kaldırmak. Eritrosit lisiz tamponu (155 mM NH4CI, 10 mM KHCO 3, 0,1mm EDTA), tek bir hücre süspansiyonu elde etmek için bir 70 mikron hücre süzgecinden filtre ve daha sonra oda sıcaklığında 10 dakika boyunca inkübe pelet tekrar. Herhangi bir yağış görülmektedir tekrar bir 70-m hücre süzgecinden Filtre. </li><li> 4 dakika boyunca 400 x g&#39;de santrifüje ve 0.5 ml PBS içinde hücre pelletini. Hücrelerin sayısını. Hücre sıralama için en az 5.000.000 hücre toplam elde edilir. SINGL sulandırmakboyama için PBS ile ml başına en az 5.000.000 hücrelere e hücre süspansiyonu. Kontrol boyamalar için 11 tüpleri (lekesiz kontrolü, negatif kontroller ve tek renkli pozitif kontroller) içine hücre süspansiyonu ve bölünmüş 50-100 ul dışarı atın. </li></ol> 3. Hücre Etiketleme ve sıralama <ol><li> Gerekirse amacı bloke edilmesi için (PBS içinde 1:10 seyreltilmiş) fare serumu 4 ℃ de 10 dakika boyunca tek bir hücre süspansiyonu inkübe edin. </li><li> Tek bir hücre süspansiyonu içine (100 tüm 1) ve 4 ℃ de 20 dakika boyunca inkübe CD34-APC, CD45, APC-Cy7, CD56-PE-Cy7, CD144 PE-ve FITC-CD146 ekleyin. Negatif kontrol için, APC- APC-Cy7-, PE-Cy7-, PE- ve FITC ile konjüge edilmiş antikor, IgG izotip eşdeğer konsantrasyonlarda ilave ediniz ve 4 ℃ de 20 dakika boyunca inkübe edin. Tek renkli Pozitif kontroller için, tek tek her bir tüp, CD34-APC, CD45, APC-Cy7, CD56-PE-Cy7, CD144 PE-ve FITC-CD146 eşdeğer konsantrasyonlarda eklenmesi ve 4 ℃ de 20 dakika boyunca inkübe edin. </li><li> İnkübasyondan sonra, 4 dakika boyunca 400 x g&#39;de santrifüj yıkayın. % 5 FBS ve% 1 P / S ilave 2 ml DMEM - 1 hücre pelletini. Hücre süspansiyonunun nihai konsantrasyonu, ml başına en az 5.000.000 hücre olmalıdır. 7-AAD (1: 100) ilave edilir ve ölü hücre çıkarılması için oda sıcaklığında 15 dakika inkübe edilir. Negatif kontrol ve tek renk ve pozitif kontrol,% 5 FBS ve% 1 P / S ile desteklenmiş 0.5 ml DMEM içinde tekrar süspansiyon hücre topaklar üretilir. </li><li> Tüpler sitometri yuvarlak alt polistiren akışına bütün hücre süspansiyonları aktarın. (Her tüp önceden doldurulmuş uygun kültür ortamı 500 ul: MECS için MPM; EGM-2 PC&#39;ler için; komisyonlarda için AC orta) hücre toplama tüpleri hazırlayın. Hücre sıralayıcı etmek için buz üzerinde Taşıma hücre süspansiyonları. </li><li> Boyanmamış kontrol, negatif kontroller, tek renkli pozitif kontroller ve ana hücre süspansiyonu sırayla hücre sıralayıcı çalıştırın hücre süspansiyonları hücre s maksimize etmek için sıkı yolluk lazer yoğunluğu, kanal tazminat ve hücre popülasyonu ayarlarkenurity (vallahi ve akış detayları için diğer dergilerde sitometri tarafından yayınlanan makalelerine bakınız). </li><li> Uygun toplama tüpleri istenilen hücre popülasyonları toplayın. 4 ℃ Mağaza, toplanan hücreler hemen ekim değilse. </li></ol> 4. Hücre Kültürü Post-sıralama <ol><li> Ile önceden kaplanmış plakalar üzerine MPM içinde &lt;10.000 hücre / cm 2 taze kriteri MECS (P0) tohum tip-I kollajen. MECS sonraki geçiş için, 3.500 tohum - ile önceden kaplanmış plakalar / matara üzerine MPM 4,000 hücre / cm 2 tip-I kollajen. </li><li> Taze% 0.2 jelatin ile kaplı plakalar üzerine EGM-2 &lt;20.000 hücre / cm 2 taze kriteri PC&#39;lerin (P0) Tohum. Bilgisayar ortamı içinde 3 oranında P2 kadar düzenli polistiren kültür levhaları üzerine aktarıldı (% 20 FBS ve% 1 P / S ile desteklenmiş DMEM): PC sonraki geçiş için, 1 hücrelerine ayrıldı. P3 yılından itibaren, tohum hücreleri 6,500 de - normal polistiren kültür plakaları / şişeleri üzerine PC ortamında 7,000 hücre / cm 2. </li><li> Normal polistiren kültür plakaları üzerine (% 20 FBS ve% 1 P / S ile desteklenmiş DMEM) ortamı içinde ac &lt;20,000 hücre / cm2 de taze kriteri ACs (P0) Tohum. Düzenli polistiren kültür plakaları / şişeleri üzerine AC ortamda 7,000 hücre / cm 2 - 6,500, sonraki komisyonlarda pasajlayarak, tohum hücreleri için. </li></ol>

<ol>
	<li>Peault, B., 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=Stem+and+Progenitor+Cells+in+Skeletal+Muscle+Development.">Stem and Progenitor Cells in Skeletal Muscle Development.</a> Maintenance, and Therapy. Mol Ther. 15, 867-877 (2007).</li><li>Toma, J. 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=Isolation+of+multipotent+adult+stem+cells+from+the+dermis+of+mammalian+skin.">Isolation of multipotent adult stem cells from the dermis of mammalian skin.</a> Nat Cell Biol. 3, 778-784 (2001).</li><li>Zuk, P. A., 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=Human+Adipose+Tissue+Is+a+Source+of+Multipotent+Stem+Cells.">Human Adipose Tissue Is a Source of Multipotent Stem Cells.</a> Mol. Biol. Cell. 13, 4279-4295 (2002).</li><li>Zimmerlin, L., 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=Stromal+vascular+progenitors+in+adult+human+adipose+tissue.">Stromal vascular progenitors in adult human adipose tissue.</a> Cytometry. 77, 22-30 (2010).</li><li>Reyes, M., 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=Origin+of+endothelial+progenitors+in+human+postnatal+bone+marrow.">Origin of endothelial progenitors in human postnatal bone marrow.</a> The Journal of Clinical Investigation. 109, 337-346 (2002).</li><li>Choi, Y., Ta, M., Atouf, F., Lumelsky, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+pancreas+generates+multipotent+stem+cells+and+pancreatic+and+nonpancreatic+progeny.">Adult pancreas generates multipotent stem cells and pancreatic and nonpancreatic progeny.</a> Stem Cells. 22, 1070-1084 (2004).</li><li>Zengin, 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=Vascular+wall+resident+progenitor+cells:+a+source+for+postnatal+vasculogenesis.">Vascular wall resident progenitor cells: a source for postnatal vasculogenesis.</a> Development. 133, 1543-1551 (2006).</li><li>Corselli, M., Chen, C. W., Crisan, M., Lazzari, L., Peault, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+ancestors+of+adult+multipotent+stem+cells.">Perivascular ancestors of adult multipotent stem cells.</a> Arterioscler Thromb Vasc Biol. 30, 1104-1109 (2010).</li><li>Ballas, C. B., Zielske, S. P., Gerson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+bone+marrow+stem+cells+for+cell+and+gene+therapies:+Implications+for+greater+use.">Adult bone marrow stem cells for cell and gene therapies: Implications for greater use.</a> Journal of Cellular Biochemistry. 85, 20-28 (2002).</li><li>Chen, C. -. W., Corselli, M., P&#233;ault, B., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Blood-Vessel-Derived+Stem+Cells+for+Tissue+Repair+and+Regeneration.">Human Blood-Vessel-Derived Stem Cells for Tissue Repair and Regeneration.</a> Journal of Biomedicine and Biotechnology. , 597439 (2012).</li><li>Zheng, B., 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=Prospective+identification+of+myogenic+endothelial+cells+in+human+skeletal+muscle.">Prospective identification of myogenic endothelial cells in human skeletal muscle.</a> Nat Biotech. 25, 1025-1034 (2007).</li><li>Okada, M., 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=Myogenic+Endothelial+Cells+Purified+From+Human+Skeletal+Muscle+Improve+Cardiac+Function+After+Transplantation+Into+Infarcted+Myocardium.">Myogenic Endothelial Cells Purified From Human Skeletal Muscle Improve Cardiac Function After Transplantation Into Infarcted Myocardium.</a> Journal of the American College of Cardiology. 52, 1869-1880 (2008).</li><li>Crisan, M., 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+Perivascular+Origin+for+Mesenchymal+Stem+Cells+in+Multiple+Human+Organs.">A Perivascular Origin for Mesenchymal Stem Cells in Multiple Human Organs.</a> Cell Stem Cell. 3, 301-313 (2008).</li><li>Park, T. S., 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=Placental+Perivascular+Cells+for+Human+Muscle+Regeneration.">Placental Perivascular Cells for Human Muscle Regeneration.</a> Stem Cells and Development. 20, 451-463 (2011).</li><li>Dellavalle, A., 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=Pericytes+of+human+skeletal+muscle+are+myogenic+precursors+distinct+from+satellite+cells.">Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.</a> Nat Cell Biol. 9, 255-267 (2007).</li><li>Dellavalle, A., 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=Pericytes+resident+in+postnatal+skeletal+muscle+differentiate+into+muscle+fibres+and+generate+satellite+cells.">Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells.</a> Nat Commun. 2, 499 (2011).</li><li>Chen, C. -. W., 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=Human+pericytes+for+ischemic+heart+repair.">Human pericytes for ischemic heart repair.</a> STEM CELLS. 31 (2), 305-316 (2012).</li><li>Corselli, M., 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=The+tunica+adventitia+of+human+arteries+and+veins+as+a+source+of+mesenchymal+stem+cells.">The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells.</a> Stem Cells and Development. 21, 1299-1308 (2012).</li><li>Campagnolo, P., 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=Human+Adult+Vena+Saphena+Contains+Perivascular+Progenitor+Cells+Endowed+With+Clonogenic+and+Proangiogenic+Potential.">Human Adult Vena Saphena Contains Perivascular Progenitor Cells Endowed With Clonogenic and Proangiogenic Potential.</a> Circulation. 121, 1735-1745 (2010).</li><li>Katare, R., 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=Transplantation+of+Human+Pericyte+Progenitor+Cells+Improves+the+Repair+of+Infarcted+Heart+Through+Activation+of+an+Angiogenic+Program+Involving+Micro-RNA-132+/+Novelty+and+Significance.">Transplantation of Human Pericyte Progenitor Cells Improves the Repair of Infarcted Heart Through Activation of an Angiogenic Program Involving Micro-RNA-132 / Novelty and Significance.</a> Circulation Research. 109, 894-906 (2011).</li><li>Zheng, B., 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=Human+myogenic+endothelial+cells+exhibit+chondrogenic+and+osteogenic+potentials+at+the+clonal+level.">Human myogenic endothelial cells exhibit chondrogenic and osteogenic potentials at the clonal level.</a> Journal of Orthopaedic Research. 31, 1089-1095 (2013).</li><li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+MSCs+Are+Pericytes.">All MSCs Are Pericytes.</a> Cell Stem Cell. 3, 229-230 (2008).</li><li>Feng, J., Mantesso, A., Sharpe, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+cells+as+mesenchymal+stem+cells.">Perivascular cells as mesenchymal stem cells.</a> Expert Opinion on Biological Therapy. 10, 1441-1451 (2010).</li><li>Tang, W., 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=White+Fat+Progenitor+Cells+Reside+in+the+Adipose+Vasculature.">White Fat Progenitor Cells Reside in the Adipose Vasculature.</a> Science. 322, 583-586 (2008).</li><li>Krautler, N. J., 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=Follicular+Dendritic+Cells+Emerge+from+Ubiquitous+Perivascular+Precursors.">Follicular Dendritic Cells Emerge from Ubiquitous Perivascular Precursors.</a> Cell. 150, 194-206 (2012).</li><li>Phinney, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+mesenchymal+stem+cells:+Implications+for+cell+therapy.">Functional heterogeneity of mesenchymal stem cells: Implications for cell therapy.</a> Journal of Cellular Biochemistry.113. 113, 2806-2812 (2012).</li><li>Chen, C. -. W., 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=Perivascular+multi-lineage+progenitor+cells+in+human+organs:+Regenerative+units,+cytokine+sources+or+both.">Perivascular multi-lineage progenitor cells in human organs: Regenerative units, cytokine sources or both.</a> Cytokine and Growth Factor Reviews. 20, 429-434 (2009).</li><li>Lin, C. -. S., Lue, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+Vascular+Stem+Cells.">Defining Vascular Stem Cells.</a> Stem Cells Dev. 22, 1018-1026 (2013).</li><li>Tang, Z., 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=Differentiation+of+multipotent+vascular+stem+cells+contributes+to+vascular+diseases.">Differentiation of multipotent vascular stem cells contributes to vascular diseases.</a> Nat Commun. 3, 875 (2012).</li><li>Zheng, B., 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+myogenic+stem+cells+from+cultures+of+cryopreserved+human+skeletal+muscle.">Isolation of myogenic stem cells from cultures of cryopreserved human skeletal muscle.</a> Cell Transplant. 21, 1087-1093 (2012).</li></ol>

J.H. received remuneration from Cook MyoSite, Inc. for consulting services and for royalties received from technology licensing during the period that the above research was performed. All other authors have no conflict of interest to disclose.

HBVSC alt popülasyonlarının belirlenmesi ve arıtma MSC ontogenezimizin anlama büyük bir ilerlemeyi temsil. MSC perivasküler kaynağını ve dokuya özel ön-madde hücreleri ve kan damarları, 22-25 arasındaki ilişkiyi gösteren gittikçe artan kanıtlar vardır. Buna ek olarak, daha fazla kapasite MSC heterojenlik ve vasküler hücre biyolojisi 26 anlaşılmasını yardımcı hBVSCs homojen bir yapıda yapıdadır izole etmek. Son birkaç yıl içinde, MECler,...

Insan iskelet kası kök / projenitör hücrelerin klinik cazip bir kaynak olarak kabul edilmiştir. İskelet kası sadece uydu hücreleri ve kas elde edilen kök hücreler (MDSCs) 1 olmak üzere miyogenik atalarıdır, iskelet miyoblastları, aynı zamanda ilkel miyogenik kök hücreleri, taahhüt değil içerir. Rejeneratif tıpta insan kas türetilmiş kök / projenitör hücrelerinin otolog veya allojenik, geniş kullanım alanı, klinik öncesi hayvan modellerinde hem de klinik çalışmalarda araştırılmıştır. Kas kök / progenitör hücrelerin rejeneratif uygulamaları kalp krizi hastalarında yaralı kalbi tamir etmek Duchenne müsküler distrofi (DMD) hastalarda distrofik kas yenileyici arasında değişir. Kemik iliğinden elde edilen multipotent yetişkin projenitör hücreler (MAPCs) ve yağ elde edilen kök hücreleri (ADSCs), yetişkin kök / dahil olmak üzere mezenşimal kök / stromal hücreleri (MSC&#39;ler) ve diğer multipotent öncü hücre popülasyonlarının keşfinden beriprogenitör hücreler yoğun tarihinden 1-9 kadar incelenmiştir. Bununla birlikte, yerinde onların yerli kimliği ve yerelleştirme retrospektif izolasyon yöntemleri örtülü durumdadır. Son zamanlarda, sıralama floresan-aktive edilmiş hücre (FACS) kullanılmasıyla, ve diğer gruplar tanımlanır ve ileriye dönük olan kan, insan iskelet kası içindeki gemiler ve çok sayıda başka organlardan izole edilebilen saf hale multipotent öncü hücre popülasyonları: kas kaynaklı endotel hücreleri (MECler), perisitlerin (PC), ve adventisyal hücreleri (Klimalar) 10. Intima tabakasında, tunika medya ve tunika adventitianın: insan kan damarı türetilmiş kök hücreler (hBVSCs) Bu üç alt-popülasyonları bulunduğunu sırasıyla kan damarlarının üç yapısal tabakalar bulunabilir. Klimalar geniş arter ve venlerin adventita tabakasında lokalize ise daha spesifik, MECler ve PC&#39;ler kılcal kılcal tespit edilir. (C MECS: Her öncül hücre alt hücre yüzey antijenleri benzersiz bir bileşimini ifadeD34 + / 56 + / 144 + / 45 -), PC&#39;ler (CD146 + / 34 - / 45 - / 56 -) ve Klimalar (CD34 + / - 31/45 - / 56 - / 146 -). Bu hBVSC altkümenin ileri karakterizasyonu üç öncü hücre popülasyonları iskelet Miyogenesis, osteogenezi, kondrogenez ve adipogenesis dahil tipik MKH&#39;lerin, benzer mezodermal gelişim potansiyelleri sahip olduğunu ortaya koydu. Tüm hBVSC alt kümeler CD44, CD73, CD90 ve CD105, taze ve kültür gibi klasik MSC belirteçler, sergilerler. Topluca bu kanıt parçaları MKH&#39;lerin damar kökeni destekledi. Ayrıca, MECS, PC&#39;ler ve komisyonlarda, terapötik kapasiteleri son zamanlarda ayrı çalışmalarda ortaya konmuştur. Yetişkin insan kas biyopsileri sıralanır MECler yaralı ve distrofik iskelet kasları ve onarım yaralı miyokardiyumu daha verimli iskelet miyoblastları ve vasküler endo yeniden gösterildiendotel hücreleri (EC). Farklı insan organlarından saflaştırılmış PC&#39;ler de onarım / yaralı ve distrofik iskelet kasları yeniden ve uydu hücre havuzu 13-16 katkıda gösterilmiştir. Çok yakın bir zamanda, insan iskelet kasından türetilmiş PC&#39;ler etkin dolaylı parakrin ve direkt hücresel etkileşimler 17 vasıtasıyla miyokard enfarktüsü tamir olduğunu göstermiştir. ACS, diğer taraftan, ya da olması ile doğrudan eksplante kan damarlarından izole edilebilir veya insan adipoz doku ve iskelet kası, FACS ile saflaştırılmıştır. Komisyonlarda, dikkate değer bir pro-anjiyojenik etkisi, fare arka kol veya bacak iskemisi-19 modelinde gösterilmiştir. Ayrıca, ACS iskemik doku tamirinde 20 ACS en güçlü terapötik potansiyelini gösteren daha etkin bir şekilde geleneksel bir MSC daha miyokard enfarktüsü onarmak için gösterilmiştir. Eşzamanlı mevcut arıtma protokolü hibe, MECler, PC&#39;ler prospektif saflaştırılması veTek bir insan iskelet kası biyopsisi vaskulatürden ACs. Bu bize çalışma ve / veya farklı terapötik amaçlar için uygun hBVSC subpopülasyonu seçim yapmanızı sağlar. Ayrıca, bu yeni teknik, ayrıca, rejeneratif tıp için multipotent öncü hücrelerin ideal bir kaynak yapma, insan iskelet kasından elde edilebilir kök / progenitör hücrelerin repertuar genişletir.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 1</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C5894</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 2</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1764</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Collagenase Type 4</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:148px;">C1889</td>
			<td style="width:193px;">Sterile vial</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD34 APC</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">555824</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD45 APC-Cy7</td>
			<td style="width:99px;">BD Pharmingen</td>
			<td style="width:148px;">557833</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD56 PE-Cy7</td>
			<td style="width:99px;">BD Pharmingen&#160;</td>
			<td style="width:148px;">557747</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Anti-human CD144 PE</td>
			<td style="width:99px;">Beckman Coulter</td>
			<td style="width:148px;">A07481</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Anti-human CD146 FITC</td>
			<td style="width:99px;">AbD Serotec</td>
			<td style="width:148px;">MCA2141F</td>
			<td style="width:193px;">Keep sterile</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">FACSAria II Flow Cytometer</td>
			<td style="width:99px;">Becton-Dickinson</td>
			<td style="width:148px;"></td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">EGM-2 Complete Medium</td>
			<td style="width:99px;">Lonza</td>
			<td style="width:148px;">CC-3162</td>
			<td style="width:193px;">For culturing PCs (only P0)</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11965</td>
			<td style="width:193px;">For culturing PCs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">DMEM high glucose (1X), liquid, with L-glutamine, with sodium pyruvate</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">11995</td>
			<td style="width:193px;">For culturing MECs and ACs</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Fetal Bovine Serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">10437-028</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Heat-inactivated horse serum</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">26050-088</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Penicillin/Streptomycin</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15140-122</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15240-062</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Trypsin-EDTA 0.5%(10X)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">15400-054</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Dulbecco&#8217;s PBS without calcium and magnesium</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:148px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Chick embryo extract</td>
			<td style="width:99px;">Accurate Chemical</td>
			<td style="width:148px;">CE650T-10</td>
			<td style="width:193px;">Filter before use</td>
		</tr>
	</tbody>
</table>

isolation of blood-vessel-derived multipotent precursors from human skeletal muscle

Mezenkimal kök / stromal hücreler (MKH) keşfinden beri, yerli kimliği ve MKH&#39;lerin yerelleştirme kültürü retrospektif izolasyon örtülü durumdadır. Son zamanlarda, floresans ile aktive edilen hücre tasnif (FACS) kullanılmasıyla, ve diğer araştırmacılar, ileriye dönük, insan iskelet kası ve damar sistemi ile bağlantılı multipotent öncü hücrelerinin üç alt-popülasyonunu tespit ve saflaştırıldı. Bu üç hücre popülasyonları: intima, media ve adventisya: Miyojenik endotel hücreleri (MECler), perisitler (PC&#39;ler) ve adventisyal hücreleri (ACs), kan damarlarının üç yapısal katmanları sırasıyla lokalize. Bu insan kan-damar kaynaklı kök hücre (hBVSC) Tüm klasik MSC işaretleri ifade ama aynı zamanda tipik MSC&#39;Iere benzer mezodermal gelişim potansiyelleri sahip nüfusun, sadece. Daha önce, MECler, PC&#39;ler ve Klimalar farklı protokoller yoluyla izole edilmiştir ve daha sonra ayrı çalışmada karakterize. Geçerli izolasyon protocol, seçici hücre yüzey belirteçleri içinde izolasyon sürecine değişiklikler ve ayarlamalar yaparak, bize aynı anda tek bir insan kas biyopsisi ile FACS tarafından üç hBVSC subpopülasyonunun arındırmak için izin verir. Bu yeni yöntem, sadece birden BVSC alt popülasyonlarının izolasyonu düzene değil, aynı zamanda farklı tedavi amaçlı hBVSCs gelecekte klinik uygulamalarını kolaylaştırmak değil.

FACS parametrelerinin ilk boyanmamış kontrol, negatif kontroller ve tek renkli pozitif kontrollerden elde edilen verilere dayalı düzeltilir. Ölü hücrelerin çıkarıldıktan sonra, floresan-etiketli hücre süspansiyonu, negatif ve pozitif hücresi yüzey işaretleyici bir dizi seçenek tabi tutulur. İlk olarak, CD45 + hücreleri, CD56 + ve CD56 önce kapılı - hücrelerin CD45 ayrılır - fraksiyonu. CD56 + CD34 +-fraksiyon CD144 seçimine tabi tutulur bur...

Watch this Scientific Journal Video about Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle at JoVE.com

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

Rejeneratif uygulamalar için idealdir insan iskelet kası liman birkaç çok-soy öncü nüfus içindeki kan damarları. Intima gelen Miyojenik endotel hücreleri, medya perisitler ve adventisyanın gelen adventisyal hücreler: Bu izolasyon yöntemi, kan damarlarının üç yapısal katmanları sırasıyla üç multipotent öncü hücre popülasyonlarının eşzamanlı arınma sağlar.

İnsan İskelet Kası Kan-damar-türevli Multipotent Öncüleri İzolasyonu

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

isolation-blood-vessel-derived-multipotent-precursors-from-human

Research

JoVE Journal

Biology

11.6K Görüntüleme.  University of Pittsburgh.  Rejeneratif uygulamalar için idealdir insan iskelet kası liman birkaç çok-soy öncü nüfus içindeki kan damarları. Intima gelen Miyojenik endotel hücreleri, medya perisitler ve adventisyanın gelen adventisyal hücreler: Bu izolasyon yöntemi, kan damarlarının üç yapısal katmanları sırasıyla üç multipotent öncü hücre popülasyonlarının eşzamanlı arınma sağlar. 

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Biyoloji

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.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

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 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.

İskelet Kası Mitokondrial İzolasyon

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

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

İskelet Kası progenitörlerin saflaştırılması

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

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

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

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

Yetişkin İskelet Kası gelen bireysel Myofibers ve Uydu Hücre İzolasyonu ve Kültür

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

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

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

Yüksek Verimli Mikroplate Solunum Ölçümler için Fare İskelet Kası Minimal Miktarları gelen Mitokondri İzolasyonu

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

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Yüksek çözünürlüklü Solunum Ölçümleri için Diferansiyel Santrifüj tarafından İskelet Kası Bozulmamış Mitokondri izolasyonu

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

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Sıçan kemik Iliği Osteoclast precursors yalıtım, arıtma ve farklılaşma

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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.

İskelet Kası Fibroblastlarından Eksozomların İzolasyonu ve Karakterizasyonu

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

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

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

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

Vibratome-Cut Miyokardiyal Dilimlerden İnsan Ventriküler Kardiyomiyositlerin İzolasyon

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