Name: analysis of pluripotent stem cells by using cryosections of embryoid bodies
Uploaded: 2010-12-08T00:00:00
Description: Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Bu çalışma Fundação de Amparo tarafından desteklenen bir Pesquisa Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ve Instituto Nacional de Ciencia e Tecnologia (INCTC). Bruna S. Paulsen ve Aline M. Fernandes EB görüntüler için minnettarız.

1. Fiksasyon ve Kriyoprezervasyon Pluripotent kök hücreleri, fare embriyonik fibroblast büyüme faktörü% 20 nakavt serum replasmanı (KSR) ve 8ng / ml (FGF-2) ile takviye mitomisin C ile inaktive edilmiş ve bakımı, DMEM/F12 içinde fibroblastlar (MEF) üzerine kültüre edildi. EB oluşumunu uyarmak için H9 hücreleri yapışmaz yemekleri, 7 gün,% 15 KSR 8 ile desteklenmiş DMEM/F12 muhafaza transfer ve kültüre edildi . Not (!): Bu teknik, herhangi bir embri...

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	<li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-117 (1998).</li><li>Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., Benvenisty, N. <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+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6, 88-95 (2000).</li><li>Kurosawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+inducing+embryoid+body+formation:+in+vitro+differentiation+system+of+embryonic+stem+cells.">Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells.</a> J Biosci Bioeng. 103, 389-398 (2007).</li><li>Conley, B. J., Young, J. C., Trounson, A. O., Mollard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+propagation+and+differentiation+of+human+embryonic+stem+cells.">Derivation propagation and differentiation of human embryonic stem cells.</a> Int J Biochem Cell Biol. 36, 555-567 (2004).</li><li>Moon, I. S., Cho, S. J., Jin, I., Walikonis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+combined+fluorescence+in+situ+hybridization+and+immunocytochemistry.">A simple method for combined fluorescence in situ hybridization and immunocytochemistry.</a> Mol Cells. 24, 76-82 (2007).</li><li>Daneshtalab, N., Dor&#233;, J. J., Smeda, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+Procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.</a> J Pharmacol Toxicol Methods. 61, 127-135 (2009).</li><li>Krenacs, L., Krenacs, T., Stelkovics, E., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval+for+immunohistochemical+reactions+in+routinely+processed+paraffin+sections.">Heat-induced antigen retrieval for immunohistochemical reactions in routinely processed paraffin sections.</a> Methods Mol Biol. , 588-5103 (2010).</li><li>Nones, J., Spohr, T. C., Furtado, D. R., Sartore, R. C., Paulsen, B. S., Guimar&#227;es, M. Z., Rehen, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+modulate+cell+survival+in+embryoid+bodies.">Cannabinoids modulate cell survival in embryoid bodies.</a> Cell Biol Int. 12, 399-408 (2010).</li><li>Huang, W. M., Gibson, S. J., Facer, P., Gu, J., Polak, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+section+adhesion+for+immunocytochemistry+using+high+molecular+weight+polymers+of+L-lysine+as+a+slide+coating.">Improved section adhesion for immunocytochemistry using high molecular weight polymers of L-lysine as a slide coating.</a> Histochemistry. 77, 275-279 (1983).</li><li>Kuenzi, M. J., Sherwood, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+specific+relaxin-binding+cells+in+thecervix,+mammary+glands,+and+nipples+of+pregnant+rats.">Immunohistochemical localization of specific relaxin-binding cells in thecervix, mammary glands, and nipples of pregnant rats.</a> Endocrinology. 136, 1367-1373 (1995).</li></ol>

Burada anlatılan yöntem sağlayan bir kolay takip PFA sabit ince Kriyostat bölümleri immünofloresan ve in situ hibridizasyon deneyleri yararlı embriyoid organları almak için protokol . Agrega olarak kendi yapısı ve organizasyonu koruyarak çıkan cryosections, insan embriyonik kök hücreleri farklılaşma hücresel ve moleküler yönlerini çalışma izin verir.

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<td>Poly-L-lysine hydrobromide</td>
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<td>Sigma</td>
<td>P2636</td>
<td>200mg/mL in water</td>
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<td>Tissue-Tek O.C.T. Compound</td>
<td>Reagent</td>
<td>Sakura</td>
<td>P2636</td>
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<td>Sucrose Solution</td>
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<td>&nbsp;</td>
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analysis of pluripotent stem cells by using cryosections of embryoid bodies

Embriyonik kök (ES) hücreleri blastosist aşamasındaki erken memeli embriyolarının 1 iç hücre kütlesinden elde edilen pluripotent hücreler. ES hücrelerinin farklılaşmasında önemli bir aşama embriyoid organlarının oluşumu (EBS), 2, 3 agrega. ES hücrelerinin yapışık olmayan plakalar kültüre EB oluşumu spontan agregasyonu üzerine dayanır. Ektoderm, mezoderm ve endoderm 4: Üç boyutlu EB özetlediği çok erken memeli embriyogenez yönlerini ve üç germ farklılaşırlar. İmmünofloresan ve in situ hibridizasyon yaygın bir doku bölüm 5, 6, 7 hücreleri hedef alan protein ve mRNA algılama teknikleri kullanılır. Burada embriyoid organlarının yüksek kaliteli cryosections oluşturmak için basit bir yöntem mevcut. Bu yaklaşım, OCT EB gömme mekansal yönelim cryosection tekniği ile takip dayanmaktadır. Ortaya çıkan bölümlerde, bazı proteinler, RNA veya DNA içeren hücreler popülasyonları için analitik yöntemler geniş bir yelpazede tabi tutulabilir. Bu anlamda, EB cryosections hazırlama (10μm) histoloji boyama analizi için gerekli araçları (örneğin Hematoxilin ve Eosin, DAPI), immünofloresan (örneğin Oct4, nestin) veya in situ hibridizasyon. Bu teknik aynı zamanda, EBS, üç boyutlu küresel yapısı bakım açısından embriyogenez yönlerini anlamak için yardımcı olabilir.

Watch this Scientific Journal Video about Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies at JoVE.com

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Pluripotent kök hücreleri süspansiyon büyüyen embriyoid organları (EBS) farklılaşırlar. Agrega olarak organizasyon korurken, embriyogenez hücresel ve moleküler yönlerini çalışmak için yararlıdır, yüksek kaliteli EB cryosections elde etmek için nasıl göstermek.

Embriyoid Kuruluşlar Cryosections kullanarak Pluripotent Kök Hücre Analizi

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

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