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4度，为以后的实验。 组装设备整个设备组装协议是biohood在无菌条件下，使用下列程序： <ol><li>清洁与图案抹下来，用70％乙醇和干燥与N 2枪的微硅主。将一个无菌的PDMS间隔1毫米的高度，围绕主的救济功能使用镊子。 </li><li>准备琼脂糖凝胶电泳在设备使用重0.3克的琼脂糖粉（尔科技）和10毫升的CO 2，独立媒体（Invitrogen公司）在50毫升的烧杯中。用抹刀和微波炉加热20秒高搅拌均匀的混合物。如果存在不溶性的颗粒，加热10秒和涡流热混合物以获得摆脱最后剩下的颗粒的混合物。重复的过程用了8秒的加热时间，然后5秒。 </li><li>快速倒到周围的硅橡胶垫片的硅主的琼脂糖溶液，并立即用无菌载玻片盖琼脂糖混合物。套用一个常数，约2分钟的幻灯片温和的压力，以确保该琼脂糖凝胶将相同的常量作为PDMS间隔1毫米的高度。 </li><li>琼脂糖凝胶电泳后，撕下图案的琼脂糖凝胶与硅橡胶垫片从硅掌握和转移到面临的幻灯片在琼脂糖凝胶微纤维连接蛋白与镀膜玻璃幻灯片。使用16号注射器尖冲水库地区的入口和出口访问的微孔。添加500μL的CO 2，独立媒体，在幻灯片上，以防止干燥的微图案的琼脂糖凝胶。 </li><li>丙烯酸多方面的设备中使用70％乙醇洗净，并用DI水冲洗。将在用琼脂糖凝胶打了个孔与微气歧管的孔。然后，将凝胶，硅橡胶垫片，纤维连接蛋白与设备的金属框架幻灯片多方面的。放入装置的螺钉，并拧紧用螺丝刀的阻力点。这样做是为了确保该设备是没有收紧，以琼脂糖微变形的程度。按顺时针顺序拧紧螺钉，以确保设备内的凝胶不移位。测试设备的泄漏和堵塞，在使用1ml注射器的微。注射器装有凝胶装枪头在管端与聚乙烯管在微注入CO 2的独立媒体。被认为是成功的媒体观察时，只从插座的微退出媒体时注入到相应的入口形成一个微。现在，该设备已准备就绪。 </li></ol>准备和播种设备中的细胞当细胞到达（大约10万细胞/ ml细胞密度）70％汇合，他们准备到器件的种子。 细胞受到两个DPBS（Invitrogen）的清洗。加入200μL胰蛋白酶- EDTA（Invitrogen）的分离细胞。细胞转移到15 mL离心管中含药血清灭活胰蛋白酶的M2媒体5毫升。离心5分钟，在1000转速细胞的生长介质。吸气关闭上清和细胞悬浮颗粒的DPBS 5毫升。在同等条件下，再次离心DPBS细胞。再次吸入关闭上清，悬浮颗粒细胞中的CO 500μL2独立媒体。 种子的细胞产生的悬浮微设备进入中心通过重力驱动流。新增的入口和出口的中心，与60μL的细胞悬液20μL的CO 2独立的中型微分别。观察中心的下一个明显微镜看细胞粘附微。这些细胞通常坚持10分钟后。细胞后坚持在一个适当的密度通道底部，pipeted在入口的剩余细胞悬液，在插座的媒体。将60μL的CO 2，独立媒体中心通道入口和出口。广场的PDMS占地面积超过日e中央入口和出口，以尽量减少蒸发媒体中心通道。 成像设备中的细胞之前，组装设备，无菌蠕动管穿过显微镜的气象站（奥林巴斯X81），和连接蠕动泵（沃森-马洛205U/CA）。通过油管冲洗，PBS在4 RPM的速度。设备的组装后，准备适当的浓度表皮生长因子（EGF）（Sigma公司）在15 mL离心管中的CO 2独立媒体的解决方案。涡EGF的解决方案，持续5秒，并与管材连接。标有相应的表皮生长因子浓度的装置连接的油管。它需要大约一个小时的EGF内传播的通道脊，并建立一个稳定的化学梯度。 Slidebook，成像软件，用来在实验运行约5小时，每10分钟的图像。 XY自动化阶段是编程的两个子领域相同的中心通道，共有三个中心通道（每个面积400μmx400μm）是在某一特定时间点的成像。在不同的中心通道（通常在一个运行3）细胞有不同的表皮生长因子的浓度，油管标记。 材料和设备从外部产后小鼠小脑6，7生发层的细胞株：C17.2细胞的神经干细胞。使用该细胞系，我们已经制定了其他两个小鼠的干细胞系，采用逆转录病毒转染法。这两个稳定转染的细胞系（1）wtEGFR - 过度表达人野生型EGFR，和（2）ΔEGFR - 这是一个组成性激活的EGFR突变。为了便于检测，逆转录病毒进行基因与核糖体结合（IRES）和绿色荧光蛋白（GFP）的内部网站的利益的骨干。 细胞培养：鼠标C17.2神经干细胞及其变种生长在M2中的媒体6孔板（10％小牛血清（FBS）（Invitrogen公司），5％马血清（HS）（Invitrogen公司的DMEM（Invitrogen公司） ），和1％青霉素/链霉素（Invitrogen公司）。）的细胞保持在37℃80％的空气和5％的CO 2 。 微流体装置：一个塑料歧管，硅橡胶垫片，不锈钢框架和设备到位。 成像一个倒置荧光显微镜（奥林巴斯IX - 81）使用，强化CCD相机（ORCA，滨松市）的连接。微流体装置被安装在自动化XYZ阶段，并保持37 摄氏度的温度环境室封闭的阶段是通常有3-4设备在同一芯片上，将在同一时间点使用自动XYZ阶段拍摄。 流量保持一个8平行线（沃森​​马洛205U/CA）自动蠕动泵是用来保持在所有的水槽和来源渠道的流动。 <img src="/files/ftp_upload/old/674_2008_9_29_6/674_Figure-01.jpg" alt="图1" width="573" height="414" /> 图1活动力三个株国科会在不同的表皮生长因子浓度（父母，wtEGFR，D表皮生长因子受体），所有的轨迹跟踪5个小时的影片（wtEGF 100ng/ml，4小时除外） ，所有曲目的由来在（0,0）重新定位，以作比较 。 <img src="/files/ftp_upload/old/674_2008_9_29_7/674_Figure-02.jpg" alt="图2" width="576" height="546" /> 图2 EGFR表达水平的相关性和活力 （一）100ng/ml表皮生长因子与微流体通道表面上的两个wtEGF神经干细胞的图像。 左边的图像是一个荧光图像，右边是一个传播的亮场图像 。 比例尺是100微米，和 通道宽度为400微米。 细胞被分为两个种群表达GFP的亮度 。 细胞 在蓝色圆圈调光器，因此较少有表皮生长因子受体的表达水平;在红色圆圈中的细胞brigher，因而有较高的表皮生长因子受体的表达水平。 （二）低和高的EGFR表达水平与细胞的轨迹。 这些轨迹是从4个小时长的电影 。

<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>
<tr>
<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>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<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>
<td>Contains 10,000 units of penicillin (base) and 10,000 g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline</td>
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<td>FBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10437-028</td>
<td>Fetal Bovine Serum, Qualified</td>
</tr>
<tr>
<td>Horse Serum</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>16050-130</td>
<td>Horse Serum, EIA tested (negative) serum</td>
</tr>
<tr>
<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>EGF</td>
<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>
<td>BP1356-500</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 ...

assessing-neural-stem-cell-motility-using-an-agarose-gel-based

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.6K 次观看.  Cornell University. 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。

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

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

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

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

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

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

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 plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

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 ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

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

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

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

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 devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

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

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 physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

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 ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.