Name: immunocytochemical analysis of human pluripotent stem cells using a self-made cytospin apparatus
Uploaded: 2010-04-09T00:00:00
Description: Watch this Scientific Journal Video about Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus at JoVE.com

<p class="jove_content">为这项工作提供部分资金由美国国家科学基金会CAREER 0744556（RAO），通过在VCU的霍华德休斯医学研究所的科学教育和研究发展计划（帕斯夸尔）的奖学金。 </p>

<p class="jove_title">第1部分：暂停免疫细胞化学染色</p><ol><li>株的细胞成单细胞悬液。 </li><li>细胞，然后转移到1.5 - 2ML离心管，离心4分钟，在200克。 </li><li>细胞被吸出上清液洗涤，重悬的PBS液1ml - （无镁<sup>2 +</sup>和Ca <sup>2 +），</sup>然后离心200克再次为4分钟。这应该是做了两次。 </li><li>洗涤后，细胞，然后重新暂停固定毫升4％多聚甲醛在室温（PFA）10分钟。 </li><li>细胞的PBS液1ml两次冲洗一遍 - 。 </li><li>洗涤后，...

<ol>
	<li>Rao, R. R., Johnson, A. V., Stice, 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=Cell+surface+markers+in+human+embryonic+stem+cells.">Cell surface markers in human embryonic stem cells.</a> <em style='font-style: italic'>Methods in Mol. Bio. - Stem Cell Assays</em>. <b>407</b>, 51-61 (2007).</li><li>Sisino, G., Bellavia, D., Corallo, M., Geraci, F., Barbieri, 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+homemade+cytospin+apparatus.">A homemade cytospin apparatus.</a> <em style='font-style: italic'>Anal. Biochem</em>. <b>359</b>, 283-284 (2006).</li></ol>

<p class="jove_content">对于我们实验室的目的，对小鼠胚胎成纤维细胞（MEFs）的干细胞成长的文化35毫米菜配发的免疫细胞化学染色程序与使用的cytospin仪器。酶传代细胞，然后收集，消除尽可能MEF层之多，成单细胞悬液。如果需要更有效地从MEF层干细胞的分离，菌落可手动挑选然后悬挂消化。如果馈线自由的条件下使用，在越来越多的干细胞培养的菌落可以简单地被刮掉，并悬挂消化。虽然最初的单细胞悬液，是理?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Lab Tek Permanox Chamber Slide</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific</td>
<td>117437</td>
<td>Material for re-used plastic slides</td>
</tr>
<tr>
<td>Superfrost/Plus Microscope Slides Precleaned</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>&nbsp;</td>
<tr>
<td>0.1-10&mu;L Filter Tips</td>
<td>&nbsp;</td>
<td>USA Scientific</td>
<td>1121-3810</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>120542-B</td>
<td>&nbsp;</td>
<tr>
<td>Cytoseal 280</td>
<td>&nbsp;</td>
<td>Richard-Allan Scientific</td>
<td>8311-4</td>
<td>Mounting Medium</td>
</tr>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Normal Goat Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10000C</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Anti-SSEA-4 Monoclonal Antibody</td>
<td>&nbsp;</td>
<td>Millipore/Chemicon</td>
<td>MAB4304</td>
<td>Primary Antibody for SSEA-4 staining</td>
</tr>
<tr>
<td>Rat Anti-SSEA-3 Monoclonal Antibody</td>
<td>&nbsp;</td>
<td>Millipore/Chemicon</td>
<td>MAB4303</td>
<td>Primary Antibody for SSEA-3 staining</td>
</tr>
<tr>
<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
<td>&nbsp;</td>
<td>Invitrogen/ Molecular Probes</td>
<td>A11029</td>
<td>Secondary Antibody for SSEA-4 staining</td>
</tr>
<tr>
<td>Alexa Fluor 488 Labeled Goat Anti-Rat IgG </td>
<td>&nbsp;</td>
<td>Invitrogen/ Molecular Probes</td>
<td>A11006</td>
<td>Secondary Antibody for SSEA-3 staining</td>
</tr>
<tr>
<td>DAPI, Dihydrochloride</td>
<td>&nbsp;</td>
<td>CalBioChem</td>
<td>268298</td>
<td>&nbsp;</td>		</tbody>
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immunocytochemical analysis of human pluripotent stem cells using a self-made cytospin apparatus

<p class="jove_content">人类多能干细胞（hPSCs），包括人类胚胎干细胞（胚胎干细胞）和人类诱导多能干细胞（人iPS细胞）是令人兴奋的细胞来源，由于其无限的自我更新能力和分化成多种细胞类型的潜力。 hPSCs多能状态是典型的评估，如定量PCR，免疫细胞化学技术，以及其他<em&gt;在体外</em&gt;<em&gt;在体内</em&gt;分化成多种细胞类型的策略。其中，免疫细胞化学技术，已发展为候选人细胞内和细胞表面的生物标志物的分析为基础的未分化状态的hPSCs的常规鉴定。鉴于hPSCs增长殖民地，问题出现在例行的基础上的单个细胞水平上量化这些标志物的表达。流式细胞仪分析服务来解决这个问题，但需要手机号码，使用的试剂通常不利于为常规质量控制评估HPSC文化。因此，发展的创建与保存完整的标记评价的单层细胞样本的实际和可重复性的手段已在干细胞研究的许多优点。这大大有利于免疫细胞化学分析，因为从单个细胞的单层，可以很方便地观察和特定标记的表达量化。实现这一目标，cytospin自制仪器构造和使用免疫细胞化学染色进行了优化。两种细胞表面标志物（SSEA3/SSEA4）的表达进行了分析，在本议定书的目的，一个变种BG01干细胞株。</p>

Watch this Scientific Journal Video about Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus at JoVE.com

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

<p class="jove_content">暂停人类多能干细胞的细胞表面标记的免疫细胞化学染色（hPSCs）（SSEA-3/SSEA-4）是实现的基础上使用自制的c​​ytospin设备创建一个观察和定量细胞单层。</p>

人多能干细胞的免疫组织化学分析，使用自制Cytospin器械

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使用胚体冰冻切片分析的多能干细胞

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Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

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To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

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人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

人类多能干细胞的神经元在一个Multititre的板格式的快速和高效地生成

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

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In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

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 ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...