Name: transfecting human neural stem cells with the amaxa nucleofector
Uploaded: 2007-07-19T00:00:00
Description: Watch this Scientific Journal Video about Transfecting Human Neural Stem Cells with the Amaxa Nucleofector at JoVE.com

<p>作者要感谢在儿童医院，奥兰治县提供hNSPC文化研究所的菲利普H ·施瓦茨博士国家人类神经干细胞资源。 </p>

<p> <strong>注：</strong>请参阅的<strong>传代人类神经干细胞</strong>的文章（ <a href="/index/Details.stp?ID=263">https://www.jove.com/index/Details.stp?ID=263</a> ）和<strong>点</strong>票<strong>人类神经干细胞</strong>的文章（ <a href="/index/Details.stp?ID=262">https://www.jove.com /指数/ Details.stp？ID = 262</a> ），以了解如何重悬并计数hNSPCs。 </p><p class="jove_step">制备</p><ol><li>确保有大量可用于转染的细胞（几百万的每个DNA的细胞转染）。 </li><li>外套的组...

<ol>
	<li>Gresch, O., Engel, F. B., Nesic, D., Tran, T. T., England, H. M., Hickman, E. S., Krner, I., Gan, L., Chen, S., Castro-Obregon, S., Hammermann, R., Wolf, J., M ller-Hartmann, H., Nix, M., Siebenkotten, G., Kraus, G., Lun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+non-viral+method+for+gene+transfer+into+primary+cells.">New non-viral method for gene transfer into primary cells.</a> <em style='font-style: italic'>Methods</em>. <b>33</b>, 151-163 (2004).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>7</b>, (2007).</li><li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Human+Neural+Stem+Cells.">Counting Human Neural Stem Cells.</a> <em style='font-style: italic'>Journal of Visualized Experiments</em>. <b>7</b>, (2007).</li></ol>

<p>这一协议描述为hNSPCs相对快速，高效的的转染过程。使用此过程中，我们已取得了初始转染效率〜35％。此过程中的转染细胞，可用于短期研究（瞬时转染的细胞）或较长期的研究中，如果选择代理是用来产生一个稳定转染的人口。我们已经产生了稳定转染的细胞在其中〜70％的细胞表达外源蛋白。在瞬时转染hNSPCs，我们所观察到的〜7天的外源蛋白的持续表达。 </p>

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<td>The Nucleofector Device</td>
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<td>Mouse neural stem cell nucleofector kit</td>
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transfecting human neural stem cells with the amaxa nucleofector

<p class="jove_content">初级哺乳动物的神经细胞，如人类神经干/前体细胞（hNSPCs）常用的阳离子脂质体转染试剂，转染，往往导致不良的细胞活力和转染效率低。其他机械方法引入一个感兴趣的基因，如基因枪或显微注射，也是有限的由差转染细胞的细胞活力和低的数字。利用病毒的结构，引入外源基因的原代细胞的战略受到了制约金额所需的时间和劳动，创建病毒载体和潜在的安全问题。我们在这里描述为转染hNSPCs使用Amaxa Nucleofector设备和技术，专门用于优化转染神经干细胞与电流的参数和缓冲溶液的一步一步的协议。使用此协议，我们已经取得了最初的转染效率〜35％和〜70％的稳定转染后。该协议需要相结合，要在适当的缓冲区Nucleofector设备电转染的DNA的hNSPCs高。</p>

Watch this Scientific Journal Video about Transfecting Human Neural Stem Cells with the Amaxa Nucleofector at JoVE.com

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

<p class="jove_content">感兴趣的基因引入细胞，为阐明其在体内的功能强大的方法。这个协议描述一个有效的方法转染人类神经干/前体细胞，使用由Amaxa Nucleofector电设备（hNSPCs）文化。</p>

人类神经干细胞转染与Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Research

JoVE Journal

Biology

Counting Human Neural Stem Cells

人类神经干细胞计数

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

科研

生物学

Passaging Human Neural Stem Cells

传代人类神经干细胞

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

免疫组化：人类神经干细胞

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

小鼠表皮神经嵴干细胞（EPI的非公务员合约雇员）文化

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Lentiviral-mediated Knockdown During <em>Ex Vivo</em> 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<em>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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

人类诱导多能干细胞的转染和Nucleofecting

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

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

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

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 ...