Name: phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors
Uploaded: 2012-07-08T14:00:00
Description: Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

我们感谢，仁泷泽伸之Onai和Markus曼茨宝贵意见。这项工作是由瑞士国家科学基金会，瑞士癌症联盟的Fondazione Ticinese每找实习SUL Cancro。

1。从骨髓细胞悬液的制备<ol><li>安乐死鼠标放置在一个不锈钢锅和喷70％的乙醇，将其腹部和背部的动物。收集后腿和胫骨股骨，脊柱。 </li><li>从脊柱准确地清除所有的软组织残留，用锋利的尖剪刀，从腿的骨头用纱布。在50毫升猎鹰管店骨头与30毫升的RPMI培养基含10％热灭活胎牛血清（FBS），血清，50μM的β-巯基乙醇，100μM的的MEM非必需氨基酸的补充，1毫米的MEM丙酮酸钠，2毫米L-谷氨酰胺，100...

<ol>
	<li>Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+units+of+development,+units+of+regeneration,+and+units+in+evolution.">Stem cells: units of development, units of regeneration, and units in evolution.</a> Cell. 100, 157-168 (2000).</li><li>Calvi, 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=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425, 841-846 (2003).</li><li>Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+haematopoietic+stem+cell+niche+and+control+of+the+niche+size.">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425, 836-841 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Arai, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie2/angiopoietin-1+signaling+regulates+hematopoietic+stem+cell+quiescence+in+the+bone+marrow+niche.">Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche.</a> Cell. 118, 149-161 (2004).</li><li>Essers, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo.">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458, 904-908 (2009).</li><li>Nagai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+on+hematopoietic+progenitor+cells+stimulate+innate+immune+system+replenishment.">Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment.</a> Immunity. 24, 801-812 (2006).</li><li>Wilson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135, 1118-1129 (2008).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273, 242-245 (1996).</li><li>Lo Celso, C., Scadden, D., D, . <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+Transplantation+of+Hematopoietic+Stem+Cells+(HSCs.">Isolation and Transplantation of Hematopoietic Stem Cells (HSCs.</a> J. Vis. Exp. (2), e157 (2007).</li><li>Romanello, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine/paracrine+stimulation+of+purinergic+receptors+in+osteoblasts:+contribution+of+vesicular+ATP+release.">Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release.</a> Biochem. Biophys. Res. Commun. 331, 1429-1438 (2005).</li><li>Casati, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+regulation+of+hematopoietic+stem+cell+cycling+activity+by+ATP.">Cell-autonomous regulation of hematopoietic stem cell cycling activity by ATP.</a> Cell Death Differ. 18, 396-404 (2011).</li><li>Bouma, G., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunological+and+genetic+basis+of+inflammatory+bowel+disease.">The immunological and genetic basis of inflammatory bowel disease.</a> Nat. Rev. Immunol. 3, 521-533 (2003).</li><li>Takizawa, H., Regoes, R. R., Boddupalli, C. S., Bonhoeffer, S., Manz, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+variation+in+cycling+of+hematopoietic+stem+cells+in+steady+state+and+inflammation.">Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.</a> J. Exp. Med. 208, 273-284 (2011).</li></ol>

这里介绍的方法能够快速，准确的分析（ 图1）个别小鼠造血。在分析各种实验设置，包括小鼠模型炎症，自身免疫性疾病，免疫缺陷，退行性疾病，代谢性疾病和癌症，允许解决的病理条件下对造血功能的影响。 图3显示了电子门李嘉诚分析细胞周期活动 CD34 -从健康小鼠的细胞和小鼠炎症性肠病（IBD）的13 Ki-67的人与生物圈计划和DAPI染色。后者...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> RPMI 1640培养</td><td> Gibco公司</td><td> 42401 </td></tr><tr><td>的MEM NEAA 100X </td><td> Gibco公司</td><td> 11140 </td></tr><tr><td>丙酮酸钠</td><td> Gibco公司</td><td> 11360 </td></tr><tr><td> PenStrep </td><td> Gibco公司</td><td> 15070 </td></tr><tr><td> PBS </td><td> Gibco公司</td><td> 20012 </td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 16000 </td></tr><tr><td>细胞过滤40微米</td><td>屋宇署猎鹰</td><td> 352340 </td></tr><tr><td> 7-AAD的染色溶液</td><td>屋宇署Pharmingen公司</td><td> 559925 </td></tr><tr><td>裂解/修复缓冲区</td><td>屋宇署Pharmingen公司</td><td> 558049 </td></tr><tr><td>烫发缓冲区III </td><td>屋宇署Pharmingen公司</td><td> 558050 </td> &lt;/ TR&gt; <tr><td> Ki-67的</td><td>屋宇署Pharmingen公司</td><td> 556026 </td></tr><tr><td>的DAPI </td><td> Invitrogen公司</td><td> D21490 </td></tr><tr><td> CD4（GK1.5） </td><td> eBioscience </td><td> 150041 </td></tr><tr><td> CD8（53-6.7） </td><td> eBioscience </td><td> 150081 </td></tr><tr><td> CD3（145-2C11） </td><td> eBioscience </td><td> 150031 </td></tr><tr><td> CD45R（，RA3-6B2） </td><td> eBioscience </td><td> 150452 </td></tr><tr><td> CD19的（6D5） </td><td> eBioscience </td><td> 150193 </td></tr><tr><td> GR1（的RB6-8C5） </td><td> eBioscience </td><td> 155931 </td></tr><tr><td> tre119（的TER-119） </td><td> eBioscience </td><td> 155921 </td></tr><tr><td> NK-1.1（PK136） </td><td> eBioscience </td><td> 455941 </td></tr><tr><td> C-包（2B8） </td><td> eBioscience </td><td> 171171 </td></tr><tr><td> （SCA-1的D7） </td><td> eBiosciENCE </td><td> 135981 </td></tr><tr><td> CD34（RAM34） </td><td> eBioscience </td><td> 110341 </td></tr><tr><td> FcγR（2.4G2） </td><td> eBioscience </td><td> 553145 </td></tr><tr><td>反装甲运兵车微珠</td><td>美天旎生物技术</td><td> 130-090-855 </td></tr><tr><td>的LS列</td><td>美天旎生物技术</td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

骨髓造血干细胞和更成熟的血细胞谱系祖居住在成年生物体和分化的主要站点。造血干细胞构成的多能干细胞能够产生所有的血细胞谱系的生命时间1分钟的细胞群。在骨髓中的造血干细胞稳态的分子解剖，具有造血，肿瘤学和再生医学的重要意义。我们描述了荧光抗体和流式细胞仪，以取得造血祖细胞亚群及个别小鼠造血干细胞的分布（ 图1）在电子门控过程的标签协议。此外，我们描述了一个方法，广泛丰富造血祖细胞，以及长期（LT）和短期（ST）的重组细胞表达c-kit的磁富集的造血干细胞从汇集的骨髓细胞悬液。由此产生的细胞制剂可用于选定的子集，并在体外进行排序在体内的功能性研究（ 图2）。 2,3都小梁成骨细胞和肝窦内皮细胞4构成支持骨髓中的造血干细胞的功能龛。几个机制中的成骨细胞的利基，包括N-cadherin的成骨细胞3子集和Tie2的表达及其配体血管生成素1 5造血干细胞的受体酪氨酸激酶的相互作用同意确定造血平静。在骨髓中的“休眠”是至关重要的，以保护造血干细胞复制和最终用尽后，过多的骑自行车活动6。外源性刺激作用于细胞的先天免疫系统，如Toll样受体配体7和α-干扰素6也将沿袭承诺祖细胞诱导的造血干细胞的增殖和分化。最近，林内的人口处于休眠状态的小鼠造血干细胞- C-KIT + SCA-1 + CD 150 + CD48 - CD34 -人口已8。细胞排序从此处所述的造血祖细胞富集的细胞悬液，CD34的表达，可以隔离两个静态的自我更新的LT-HSCs和ST-造血干细胞9。一个类似的过程基于血统阳性细胞的枯竭和LT-HSC的排序与CD48和Flk2抗体的先前已描述10。在本报告中，我们提供了一个协议表型特征和体外造血祖细胞，它可以用于监测在不同的生理和病理条件下的造血细胞周期分析。此外，我们描述了流式细胞仪分选造血干细胞的过程，它可以用来定义调节他们的自我更新，在细胞生物学和信号转导检测，以及用于移植的扩张和分化的因素和机制。

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

描述的方法来分析流式细胞仪检测骨髓造血祖细胞的分布以及有效地隔离高度纯化造血干细胞（HSCs）。基本上是基于C-kit +细胞和排序，以净化造血干细胞和分子生物学研究细胞的磁富集分离过程。

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Research

JoVE Journal

Biology

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

小鼠卵黄囊和AGM早期造血干细胞的分离

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

分离，培养和移植肌卫星细胞

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Isolation of Murine Valve Endothelial Cells

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

小鼠胚胎的逆转录病毒感染干细胞衍生胚体细胞造血分化的分析

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

在EML造血前体细胞调节自我更新和分化的关键因素，通过RNA测序分析鉴定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...