Name: assessing neural stem cell motility using an agarose gel-based microfluidic device
Uploaded: 2008-02-11T02:20:00
Description: Watch this Scientific Journal Video about Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device at JoVE.com

支持这项工作是由纽约州厅科学，技术和学术研究（NYSTAR）（在一个先进的技术授予中心的形式），纳米生物技术中心（NBTC），一个国家的STC方案补助根据协议ECS - 9876771号科学基金会，美国脑肿瘤协会和纽约香港医学专科学院（申诉）。

程序与纤维连接蛋白涂层的幻灯片微流体装置组装之前，与纤维连接蛋白消毒玻片。外套biohood幻灯片保持无菌。沿幻灯片的边缘对齐无菌硅橡胶垫片和侧面标记，正面临着一个永久性标记。吸取1毫升5微克/ ml的溶液在载玻片上的全部硅橡胶垫片内的纤维连接蛋白（Sigma公司）。离开了一个小时的幻灯片不受干扰，然后完全干燥使用的N 2枪。幻灯片可以储存在摄氏...

<ol>
	<li>Blow, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+in+search+of+a+killer+application.">Microfluidics: in search of a killer application.</a> Nature Methods. 4, 665-670 (2007).</li><li>Shing-Yi Cheng, S. H., Wassweman, M. a. x., Archer, S. h. i. v. a. u. n., Shuler, M. i. c. h. a. e. l. . L., Wu, M. i. n. g. m. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hydrogel-based+microfluidic+device+for+the+studies+of+directed+cell+migration.">A hydrogel-based microfluidic device for the studies of directed cell migration.</a> Lab-on-a-chip. 7, 763-769 (2007).</li><li>Pedersen, M. W., Tkach, V., Pedersen, N., Berezin, V., Poulsen, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+naturally+occurring+constitutively+active+variant+of+the+epidermal+growth+factor+receptor+in+mouse+fibroblasts+increases+motility.">Expression of a naturally occurring constitutively active variant of the epidermal growth factor receptor in mouse fibroblasts increases motility.</a> Int J Cancer. 108, 643-653 (2004).</li><li>Ayuso-Sacido, A., Graham, C., Greenfield, J. P., Boockvar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+duality+of+EGFR+signaling+and+neural+stem+cell+phenotype:+Cell+enhancer+or+cell+transformer.">The duality of EGFR signaling and neural stem cell phenotype: Cell enhancer or cell transformer.</a> Current Stem Cell Research and Therapy. 1, 231-238 (2006).</li><li>Eisenbach, M., Constantinou, C., Aloni, H., Shinitzky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repellents+for+Escherichia+coli+operate+neither+by+changing+membrane+fluidity+nor+by+being+sensed+by+periplasmic+receptors+during+chemotaxis.">Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis.</a> J Bacteriol. 172, 5218-5224 (1990).</li><li>Snyder, E. Y., Deitcher, D. L., Walsh, C., Arnold-Aldea, S., Hartwieg, E. A., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+neural+cell+lines+can+engraft+and+participate+in+development+of+mouse+cerebellum.">Multipotent neural cell lines can engraft and participate in development of mouse cerebellum.</a> Cell. 68, 33-51 (1992).</li><li>Snyder, E. Y., Yoon, C., Flax, J. D., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+neural+precursors+can+differentiate+toward+replacement+of+neurons+undergoing+targeted+apoptotic+degeneration+in+adult+mouse+neocortex.">Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.</a> Proc Natl Acad Sci U S A. 94, 11663-11668 (1997).</li></ol>

图1显示了三个国科会菌株，亲代细胞，wtEGFR和ΔEGFR（控制）的轨迹。每个设定的轨迹，从5个小时长的电影。父母的细胞，细胞运动达到顶峰时，表皮生长因子的浓度是〜在10ng/ml; wtEGFR细胞，细胞的运动高峰期转移到100ng/ml或以上;ΔEGFR细胞，细胞运动是独立的表皮生长因子浓度如预期。 与100ng/ml wtEGF细胞最能动的细胞，他们移动了约1.5倍的速度比用在10ng/ml亲代细胞。这表明：（1）对EGFR的表达，增强细胞活?...

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<td>Dulbecco's Modified Eagle Medium (DMEM) (1X)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>11971-025</td>
<td>&nbsp;</td>
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<td>CO2-independent media</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>18045-088</td>
<td>A non-HEPES proprietary medium suitable for supporting cell growth for a variety of epithelial, fibroblast, and lymphoid cell lines in atmospheric conditions, contains no L-glutamine</td>
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<td>Trypsin-EDTA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25200-072</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14190-144</td>
<td>Phosphate-buffered saline, without calcium or magnesium</td>
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<td>Pen/Strep</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
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<td>Invitrogen</td>
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<td>Fetal Bovine Serum, Qualified</td>
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<td>Horse Serum</td>
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<td>Invitrogen</td>
<td>16050-130</td>
<td>Horse Serum, EIA tested (negative) serum</td>
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<td>Fibronectin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0895</td>
<td>Fibronectin from human plasma, also called cold insoluble globulin</td>
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<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>E4127</td>
<td>Epidermal Growth Factor from murine submaxillary gland, lyophilized</td>
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<td>Agarose</td>
<td>Reagent</td>
<td>FisherScientific</td>
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assessing neural stem cell motility using an agarose gel-based microfluidic device

微流体技术是成熟的一个新的水平，达到大分子检测细胞为基础的分析是仍处于起步阶段 1 。这主要是由于哪一个可以创建一个细胞的兼容和稳定的微环境，使用传统的微细加工技术和材料的困难。我们解决这个问题，通过引入一种新型的微细加工的材料，琼脂糖凝胶作为基体材料的微流体装置。琼脂糖凝胶是高度的可塑性，并渗透到细胞存活所必需的气体和营养成分，从而细胞检测的理想材料。我们已经表明以前，琼脂糖凝胶为基础的设备已成功在学习细菌和中性粒细胞迁移2。在这份报告中，三个平行的微流体通道，在一个约1mm厚度的琼脂糖凝胶膜图案。与媒体/缓冲区的恒定流量均保持在两个侧面采用蠕动泵渠道。细胞均保持在观察的中心通道。由于营养物质和化学品在一旁渠道不断扩散的化学环境的中心通道的中心通道，从侧面很容易通过沿侧渠道流控制。使用这种设备，我们表明，神经干细胞的运动，可以监视各种化学条件下轻松光学，和实验结果表明，表皮生长因子受体（EGFR）的过度表达，增强了神经干细胞的活力。神经干细胞的运动功能是一个重要的生物标志物的评估细胞的侵略性，从 ​​而致瘤因素 3 。解读国科会蠕动的内在机制，将产生的神经系统发育障碍和洞察到脑肿瘤干细胞的侵袭。

Watch this Scientific Journal Video about Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device at JoVE.com

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

我们表明，表皮生长因子受体（EGFR）的过度表达，增强了神经干细胞使用一种新型的琼脂糖凝胶微设备（神经干细胞）的活力。这项技术可以很容易地适应其他哺乳动物细胞系统细胞来源匮乏，如人类神经干细胞，的，和周围的时间又是至关重要的的。

使用琼脂糖凝胶为基础的微流体装置的评估神经干细胞的运动

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

Research

JoVE Journal

Biology

Culture of Mouse Neural Stem Cell Precursors

培养小鼠神经干细胞的前体

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

科研

生物学

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

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 ...

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 ...

A Gradient-generating Microfluidic Device for Cell Biology

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Cell Capture Using a Microfluidic Device

细胞捕获使用一个微流体装置

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

PDMS微流体装置的廉价制造的非等离子键合

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

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 ...