前列腺细胞系敬请凌钰王博士和幸吉恩孔博士在癌症中心，加州大学戴维斯分校提供的。该项目由美国国立卫生研究院资助趋电性支持4R33AI080604。

1.培养前列腺细胞<ol><li>培养PRNS-1-1和PNT2前列腺细胞上100毫米培养皿在RPMI 1640培养基补充有10％FBS和抗生素-抗真菌剂，在37℃，5％的CO 2。每天刷新培养基，直到细胞达到80％汇合的趋电实验。 </li></ol> 2.装配趋电钱伯斯<ol><li>组装底部室<ol><li>擦拭塑料趋电室用2-丙醇。适用的海洋级硅密封剂周围的圆形开口与注射器的腔室的底侧，并附加一个大玻璃罩（ 图1）。采用棉花施用器的背面侧，以向下推动玻璃罩和擦去多余胶用棉签。所述圆形开口允许介质和电流流过位于这两个内层介质储层之间的细胞。 </li><li>切一小玻璃罩，使的6毫米x 25毫米垫片用钻石点标记。 </li><li>翻转室。应用2条纹的硅胶，用注射器和/或金属刮刀以创建为2圆形开口，底部玻璃之间细胞附着以10mm×25mm的通道表面。胶2片玻璃间隔2个圆孔之间。使用棉涂敷器的背面侧，以向下推隔板和擦去多余胶用棉签和/或牙签。 </li><li>干燥室24小时，直至在硅胶固化。浸泡室在蒸馏水过夜以从胶除去乙酸残基。干燥室供立即使用，或在腔室，供以后使用存储在一个干净的容器中。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig1highres.jpg" width="500" /> 图1:底部趋电室的组装。 A）大玻璃罩附着到清洁室中。B的底部）TWO玻璃衬垫片的2圆形开口之间胶合以创建为10mm×25mm的通道供细胞附着。 </li></ol></li><li>在趋电室种子细胞<ol><li>准备实验所需趋电室的要求数量。每个细胞系或治疗需要一个单一的趋电室中。擦拭趋电室与2-丙醇。用无菌PBS 3次洗室并检查室2个圆孔之间的液体流动。 </li><li>括在无菌细胞培养皿的腔室，并允许他们平衡在37℃下进行15分钟。分离从用5毫升0.25％胰蛋白酶-EDTA的培养皿的前列腺细胞在37℃下5分钟。中和胰蛋白酶-EDTA用PBS5毫升10％FBS中。 </li><li>转移细胞到15ml管，并在200×g离心，37℃离心细胞5分钟。吸出上清液并重新悬浮细胞于1ml培养基中。 </li><li>就拿20μ升的细胞溶液加载到一个计数室并计算细胞数。调节细胞浓度至8×10 4个细胞/毫升培养基。 </li><li>就拿趋电室出37℃的孵化器。种子350微升细胞悬浮液在每个室中。 </li><li>在培养皿用湿的Kimwipe或在湿度室中在培养箱中在37℃，5％CO 2孵育过夜，在室中的细胞。 </li></ol></li></ol><img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig2highres.jpg" width="500" /> 图2:接种细胞到所述腔室的底部室中干燥并清洁A）的前列腺细胞胰蛋白酶处理，计数并传送到所述腔室并孵育过夜B）中的顶玻璃罩附着拍摄前，以密封腔室。 <ol start="3"><li>一ssembling顶部室<ol><li>组装前拍摄前室。显微镜只能容纳成像单个趋电室在同一时间。预热10毫升培养基在37℃下为每个趋电室中。转让从孵化器趋电室，以37℃加热板。 </li><li>冲洗细胞培养基以除去未附着的细胞。离开400微升培养基中的腔室中。用注射器施加高真空润滑脂在两个玻璃间隔件的顶部。 </li><li>添加一个小玻璃罩密封室。轻轻向下压用棉涂敷器的背面侧的玻璃罩。擦去多余的介质棉签。 </li><li>干燥的玻璃表面，并应用在高真空油脂来密封玻璃罩和腔室之间的间隙。使用金​​属铲传播的油脂。加入4毫升培养基中于内水库和检查储层之间的液体流动。 </li></ol></li><li>使琼脂桥梁<ol><li>切一对2英寸长的PVC管（一十六分之五百零三ID X 5/16外径x 1/16墙），翻转并将其插入到100毫升的烧杯中。 </li><li>测量200毫克细菌用琼脂，并将其加入，在50毫升烧瓶中，用10ml培养基，使2％的琼脂凝胶。微波30秒。加载琼脂凝胶的塑料管具有一个移液管。离开琼脂桥在室温下10分钟以固化该琼脂。 </li><li>加2ml培养基的趋电室的外储层。插入琼脂桥的内部和外部介质油藏进行的电流。 </li></ol></li></ol><img alt="图3" fo:content-width="3.5in" src="/files/ftp_upload/51973/51973fig3highres.jpg" width="350" /> 图3:拍摄上腔室中的细胞的图示来演示趋电室的最后的组装。该腔室是完全密封的高真空润滑脂。介质被加入叔Ó填补水库，和两个琼脂桥被插入到腔室中。然后将趋电室传送到显微镜阶段和电极连接到施加电场。侧视组装趋电室的被示出为证明该电流通过琼脂桥和盖玻璃之间的空间流过的细胞。 3.时间推移​​成像<ol><li>在环境室切换到37℃，用5％的CO 2中的空气循环2小时之前成像。 </li><li>打开显微镜和启动图像采集软件。校准显微镜阶段，并选择10X物镜。 </li><li>转移趋电室的显微镜阶段，确保室用胶带和专注于细胞。插入趋电电极到外储层阴极右侧。固定电线用胶带的电极。 </li><li>开关电源博x到电场施加到腔室中。测量的输出电压与整个室中的电压表，达到2.5伏为25mm长的腔室（100毫伏/毫米）。通过调节输出电流保持在实验过程中的场强。 </li><li>选择跨室10分的软件，电影，产生10定时短片。设置获取条件以10分钟的间隔为2小时。 </li><li>开始拍摄，并根据需要调整输出电流。 </li><li>在实验结束时，取出从舞台的腔和固定细胞用95％的酒精。破开了腔用刀片清理和重新使用它。 </li></ol> 4.量化趋电性的<ol><li>旋转时间推移电影和重新定位阴极到的图像的顶部。导出电影的单元格跟踪软件。 </li><li>手动跟踪（X，Y）10-20细胞在每个时间点从每部电影（ 图4 A，B）的位置。相对于南北方向上的迁移距离和角度，电场的方向相同，将计算在小区的跟踪软件（ 图4C）。 注:平均迁移速度被从总迁移距离和总拍摄时间换算。方向性是从垂直于余弦值进行转换。如果细胞随机方向性迁移，净余弦平均将接近零。如果细胞直接迁移向阴极，余弦值将是1。如果细胞直接迁移向阳极，余弦值将是-1。 </li></ol><img alt="图4" fo:content-width="4in" src="/files/ftp_upload/51973/51973fig4highres.jpg" width="400" /> 图4:细胞跟踪测量方向性跟踪线细胞图像A）覆盖。细胞的（X，Y）位置是manuaLLY跟踪在时间推移的电影。如果细胞迁移随机，平均余弦接近于零B）但是，如果细胞向阴极或阳极迁移的平均余弦的值是接近+（阴极）或- （阳极）1.0C 的 ）的方向性，提出通过余弦值，这是从迁移的角度（θ）进行转换。余弦（θ）等于距离a（迁移距离），以距离B（投影于电场的方向上的距离）的比率。 <ol start="3"><li>保存测量结果。导入数据到数据库应用程序来计算组合的结果。 </li><li>平均迁移速度和平均余弦值组合的数据导出到电子表格绘制棒图（ 图5）。 </li></ol>

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(68), (2012).</li><li>Meng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-controlled+directed+migration+of+neural+progenitor+cells+in+2D+and+3D+environments.">Electric field-controlled directed migration of neural progenitor cells in 2D and 3D environments.</a> J Vis Exp. (60), (2012).</li><li>Pullar, C. E., Isseroff, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+mediates+keratinocyte+directional+migration+in+an+electric+field.">Cyclic AMP mediates keratinocyte directional migration in an electric field.</a> J Cell Sci. 118 (9), 2023-2034 (2005).</li><li>Sheridan, D. M., Isseroff, R. R., Nuccitelli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imposition+of+a+physiologic+DC+electric+field+alters+the+migratory+response+of+human+keratinocytes+on+extracellular+matrix+molecules.">Imposition of a physiologic DC electric field alters the migratory response of human keratinocytes on extracellular matrix molecules.</a> J Invest Dermatol. 106 (4), 642-646 (1996).</li><li>Feng, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+migration+of+neural+stem+cells+derived+from+human+embryonic+stem+cells+by+an+electric+field.">Guided migration of neural stem cells derived from human embryonic stem cells by an electric field.</a> Stem Cells. 30 (2), 349-355 (2012).</li><li>Mukerjee, E. V., Isseroff, R. R., Nuccitelli, R., Collins, S. D., Smith, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microneedle+array+for+measuring+wound+generated+electric+fields.">Microneedle array for measuring wound generated electric fields.</a> Conf Proc IEEE Eng Med Biol Soc. 1, 4326-4328 (2006).</li><li>Nuccitelli, R., Nuccitelli, P., Li, C., Narsing, S., Pariser, D. M., Lui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electric+field+near+human+skin+wounds+declines+with+age+and+provides+a+noninvasive+indicator+of+wound+healing.">The electric field near human skin wounds declines with age and provides a noninvasive indicator of wound healing.</a> Wound Rep. and Reg. 19, 645-655 (2011).</li></ol>

The authors have nothing to disclose.

一个细胞的趋电性反应的分析，一直是许多细胞迁徙或增长的重要功能指标处理27，28。在这里，我们使用一个特制室玻璃表面拍摄2前列腺细胞系。这些细胞系表现出不同程度的趋电的，和我们推测，细胞内定位或趋电介导蛋白的激活可能产生不死的细胞系，从而导致在趋电响应的观察到的差异的过程中受到干扰。 相比在培养皿27，28作出其它趋电室的设计中，有几个好处，以目前的设计。首先，将玻璃表面适于成像，并与细胞中的玻璃罩可以趋电对于免疫染色后进行检索。在这项研究中，我们使用的10X物镜低倍率来监视一组细胞。然而，随着THESË改进玻璃光学，所以能够拍摄单个细胞以较高的放大倍率用60X油浸物镜，用于跟踪快速扩展lamellipodial的和缩回的单元17的前缘，或跟踪标记的细胞的荧光蛋白质。第二，在玻璃表面可以被改性基质蛋白涂层10,12或图案化的微通道。化学处理，也可以预先拍摄29日前处理细胞。不需要在这里所用的前列腺细胞系基质涂层，基质涂层是细胞类型依赖性的。例如，I型胶原涂层所需的人角质形成细胞30的趋电，但层粘 ​​连蛋白涂层所需的人神经干细胞31的趋电。我们在胶原I，胶原蛋白IV，纤连蛋白，层粘连蛋白和测试人角质细胞趋电。不同的基质蛋白改变细胞的迁移速度，但并没有改变阴极migrati<html>在趋电29。第三，改性铂电极是惰性的电场。相比，与其他趋电方法27中使用的银电极，它们不分解或释放出金属离子的介质，从而降低了细胞毒性趋电。第四，我们可能会改变帧捕捉，这有助于我们监测快速或慢速细胞迁移的时间间隔。该电动显微镜也提供了机会，获得来自不同领域的多个电影在一个腔。的对照实验没有施加电场是用于确定是否所述电场改变了细胞的迁移速度非常重要的。 然而，目前的趋电室被限制为拍摄的细胞迁移对于长达4小时，由于在储层小体积培养基。用的腔室设计的放大版本，这将是可能的薄膜电池的时间较长。 <html> ve_content"&gt;在该方法的一些潜在的问题包括:1）的细胞可能在腔室组装期间死如果它们变得干涸由于在所述腔室（400微升）非常小的体积的介质中，它避免捕集空气是很重要的气泡在腔室，并保持一些液体在细胞漂洗步骤覆盖细胞。2）的腔室可以在成像期间泄漏，这将降低电流通过腔室的液体流在腔室应仔细检查它之前转移到显微镜载物台和更高真空润滑脂施加如果需要的话。3）出现模糊的高真空润滑脂可以与拍摄干扰的玻璃表面可以用95％酒精清洗以去除油污。4）阶段漂移或腔室上的任意位移一个电动显微镜阶段可能偏压定向迁移的测量结果。因此，为了确保趋电室提供足够的阶段保持器和标签的磁带是非常重要的。 ntent"&gt;总的来说，趋电的方法是有用的，以揭示游动细胞的固有方向性在施加电场，这将有助于我们进一步了解在生物体的定向迁移。

内源性电场在各种组织，如皮肤1，32，33和脑2检测到。生理电场供应特定的生物功能，包括引导胚胎发育3，第4，指导神经元突起5,6的向外生长，促进上皮细胞和角膜伤口闭合1,7。 在体外 ，施加直流电场来培养细胞模仿生理电场和诱导定向细胞迁移，或趋电。趋电已经研究中的成纤维细胞8，鱼角化9，人上皮和角膜角化10-12，淋巴细胞13，神经母细胞2和神经元祖细胞14。当暴露于施加的场，大部分研究细胞的定向迁移朝向阴极（ - ）极。然而，一些癌症的细胞，包括高转移人乳腺癌细胞和人前列腺癌细胞系PC-3M，移动到anodal（+）极15,16。提出调解趋电或解释的细胞，以感测电场的能力，包括活化几种机制的表皮生长因子受体12，上皮钠通道17，PI3K和PTEN的18，和钙离子释放15，19，其机制还没有完全理解，这是可能的，多种信号传导途径中涉及趋电。 表征粘附，游动细胞的定向迁移，既可以监视单个细胞迁移10，12，17或迁移融合细胞的片材18，20，该技术是由改性后，我们在这里展示的2维趋电的方法是有用鹏贾菲21，和西村等 10与定制，透明PVC室，可移动coverslIPS允许趋电性进行二次分析，如免疫荧光成像后容易细胞检索。的趋电室中的玻璃表面的光兼容，这使得拍摄在高放大倍率和与荧光标记的细胞。它也允许在实验设计与玻璃表面的改性，如改变表面涂层或收费。制成的第1号盖玻片隔件被用于在腔室，以最小化电流的流动过的细胞;因此焦耳热，这是成比例的电流的平方，将不会在实验过程中过热的细胞。连接琼脂桥防止与细胞中的电极的直接接触，并防止在趋电介质的pH值或离子浓度的改变。 两个非致瘤性的人类前列腺细胞系检查在这项研究中的趋电响应。该PRNS-1-1 22和PNT223顷既SV40永生化，生长因子依赖性细胞系的表达上皮标志物细胞角蛋白5，8，18和19与前列腺特异性抗原（PSA）的低或无表达。两种细胞系维持正常上皮细胞的多边形形态，但染色体异常中观察到核型22，24，虽然PRNS-1-1和PNT2在大多数实验中有着相似的行为，但它们确实显示出在腺泡结构的形成，并在不同趋电性。在3-D矩阵，基底膜，将PRNS-1-1细胞形成空心腺泡结构，类似于流明的正常前列腺腺体组织25。然而，PNT2细胞形成固体球体无管腔或极化上皮26。所述PRNS-1-1细胞也表现出比在目前的研究中PNT2更高趋电响应。形成腺泡结构和趋电之间的相关性PRNS-1-1表明趋电信号可在组织PR发挥作用ostate腺组织的运动响应于内源性电场，并提供了进一步的特征，这些2细胞系之间进行区分。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Cells&lt;/strong&gt;</td></tr><tr><td>pRNS-1-1 prostate cells</td><td></td><td></td><td>Lee, &lt;em&gt;et al.&lt;/em&gt; (1994)</td></tr><tr><td>PNT2 prostate cells</td><td>Sigma-Aldrich</td><td>95012613-1VL</td><td>Berthon, &lt;em&gt;et al.&lt;/em&gt; (1995)</td></tr><tr><td colspan="4">&lt;strong&gt;Medium and solutions&lt;/strong&gt;</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Invitrogen</td><td>11875-093</td><td>warm up to 37 &amp;deg;C before use</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>10% in PBS, warm up to 37 &amp;deg;C before use</td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Life Technologies</td><td>15240</td><td>add 5 ml to 500 ml medium</td></tr><tr><td>2-propanol</td><td>VWR</td><td>BDH1133-5GL</td><td></td></tr><tr><td>PBS</td><td>-</td><td>-</td><td>137 mM NaCl, 2.7 mM KCl, 4.3 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.5 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; in 1,000 ml of H&lt;sub&gt;2&lt;/sub&gt;O, pH to 7.4 and autoclaved, warm up to 37 &amp;deg;C before use</td></tr><tr><td>0.25% Trypsin-EDTA&amp;nbsp;</td><td>Invitrogen</td><td>25200-056</td><td>warm up to 37 &amp;deg;C before use, treat cells for 3-5 min at 37 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;Galvanotaxis device&lt;/strong&gt;</td></tr><tr><td>Galvanotaxis chambers</td><td>Precision Plastics Inc, CA</td><td></td><td>Custom-designed (1/4&quot; x 2&quot; x 3.5&quot;), non-toxic, clear PVC chambers. Please contact the authors for the design specs.</td></tr><tr><td>Galvanotaxis electrodes</td><td>UCD electric shop</td><td></td><td>platinum coiled electrodes with flexable cords</td></tr><tr><td>Galvanotaxis power box</td><td>Substrate Engineering, CA</td><td></td><td>custom-designed DC power output with voltmeter</td></tr><tr><td>Microscope Cover Glass, Large, 45 x 50 mm, No. 1.5</td><td>Fisher</td><td>12-544-F</td><td></td></tr><tr><td>Microscope Cover Glass, small, 25 x 25 mm, No. 1</td><td>ThermoScientific</td><td>3307</td><td></td></tr><tr><td>Diamond point marker</td><td>ThermoScientific</td><td>750</td><td></td></tr><tr><td>Marine grade silicon sealer, clear</td><td>3M</td><td>051135-08019</td><td></td></tr><tr><td>High vacuum grease</td><td>Dow Corning</td><td>2021846-0807</td><td></td></tr><tr><td>6 ml syringe</td><td>Fisher Scientific</td><td>05-561-64</td><td></td></tr><tr><td>Nichiryo Syringe, 1.5 ml</td><td>Nichiryo</td><td>SG-M</td><td></td></tr><tr><td>Cotton applicators</td><td>Purtian Medical Products</td><td>806-WC</td><td></td></tr><tr><td>Qtips</td><td>Johnson &amp;amp; Johnson</td><td>729389</td><td></td></tr><tr><td>Nalgene 180 PVC tubing&amp;nbsp;</td><td>Nalgene</td><td>8000-9030</td><td>503/16 ID x 5/16 OD x 1/16 Wall</td></tr><tr><td>Bacto-Agar</td><td>Difco</td><td>0140-01</td><td>make 2% agar solution</td></tr><tr><td>Razor Blade</td><td>Personna</td><td>74-0001</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipments and Software&lt;/strong&gt;</td></tr><tr><td>Benchtop Centrifuge</td><td>Eppendorf</td><td>5810R</td><td>operated with an A-4-62 rotor</td></tr><tr><td>Cellometer Auto T4</td><td>Nexcelom</td><td>Auto T4</td><td></td></tr><tr><td>Cellometer counting chambers</td><td>Nexcelom</td><td>CHT4-SD100-002</td><td>load 20 &amp;#956;l cell solutions to count</td></tr><tr><td>Culture Temp Warming plate</td><td>Bel-Art Scienceware</td><td>370150000</td><td>to keep the galvanotaxis chambers at 37 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Eclipse TE-2000 microscope with motorized stage and environmental chamber</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor 10X/0.30 objective len</td><td>Nikon</td><td></td><td></td></tr><tr><td>Retiga EX CCD camera&amp;nbsp;</td><td>Qimaging</td><td></td><td>Cooled CCD camara, mono-color, 12-bit</td></tr><tr><td>Compressed air with 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Airgas</td><td></td><td>special order</td></tr><tr><td>Volocity 6.3</td><td>PerkinElmer</td><td></td><td>Image acquiring software</td></tr><tr><td>Improvision OpenLab 5.5.2</td><td>PerkinElmer</td><td></td><td>Cell tracking software and customized to measure migration angles</td></tr><tr><td>FileMaker Pro Advanced, 8.0</td><td>FileMaker</td><td></td><td></td></tr><tr><td>Microsoft Excel 2008 for Mac</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells

生理电场供应特定的生物功能，如指导细胞迁​​移在胚胎发育，神经元向外生长和上皮伤口愈合。 体外施加直流电场到培养的细胞诱导定向细胞迁移，或趋电。我们在这里展示的2维趋电方法经修改以定做的聚（氯乙烯）（PVC）的腔室，玻璃表面，铂电极和使用在其上的细胞成像的机动阶段。在PVC室和铂电极具有低毒性，且价格合理和可重复使用的。在玻璃表面和电动显微镜阶段改善图象的质量，并允许可能的修改，以在玻璃表面，并处理该细胞。我们拍摄的两个非致瘤性，SV40永生化前列腺细胞系，PRNS-1-1和PNT2的趋电。这两种细胞系表现出类似的迁移速度和迁移均朝阴极，但它们确实显示出不同程度的方向性的趋电。通过此协议中得到的结果表明，PRNS-1-1和PNT2细​​胞株可具有管其定向洄游响应不同的内在特征。

两行前列腺细胞（PRNS-1-1和PNT2）进行了调查用这种方法。在这两个细胞系在超过2小时（ 图5A）的过程类似的速度为1.0 +/- 0.3微米/分钟迁移。然而，方向性的电场是0.7 +/- 0.3的PRNS-1-1线，和0.2±0.8对PNT2线（ 图5B）。结果显示在这两个细胞系的趋电一显著差异（p &lt;0.01，100个细胞被跟踪），这表明它们具有导致不同的定向响应位置线索不同细胞信号传导机制。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51973/51973fig5highres.jpg" width="500" /> 趋电前列腺细胞:图5。 A）中 PRNS-1-1和PNT2线的迁移速度是相似当细胞暴露于电场。乙 ）然而，PRNS-1-1线显示出高方向性朝向阴极（余弦= 0.7），而PNT2线呈低方向性（余弦= 0.2）中的电场。

Watch this Scientific Journal Video about Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells at JoVE.com

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

利用定制设计的趋电钱伯斯研究前列腺细胞定向迁移

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

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9.1K 次观看.  University of California, Davis. We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

视频: Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed.

Here we explain a protocol for modelling the biophysical microenvironment where crosslinking and increased stiffness of the basement membrane (BM) induced by advanced glycation endproducts (AGEs) has pathological relevance.

如何学习基底膜刚度生物物理触发在前列腺癌和其他与年龄相关的病理或代谢性疾病

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

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癌症研究

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

生物工程

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

医学

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

在神经节尊贵的文化产生细胞的迁徙行为

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

神经科学

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

A Galvanotaxis Assay for Analysis of Neural Precursor Cell Migration Kinetics in an Externally Applied Direct Current Electric Field

The discovery of neural stem and progenitor cells (collectively termed neural precursor cells) (NPCs) in the adult mammalian brain has led to a body of research aimed at utilizing the multipotent and proliferative properties of these cells for the development of neuroregenerative strategies. A critical step for the success of such strategies is the mobilization of NPCs toward a lesion site following exogenous transplantation or to enhance the response of the endogenous precursors that are found in the periventricular region of the CNS. Accordingly, it is essential to understand the mechanisms that promote, guide, and enhance NPC migration. Our work focuses on the utilization of direct current electric fields (dcEFs) to promote and direct NPC migration - a phenomenon known as galvanotaxis. Endogenous physiological electric fields function as critical cues for cell migration during normal development and wound repair. Pharmacological disruption of the trans-neural tube potential in axolotl embryos causes severe developmental malformations1. In the context of wound healing, the rate of repair of wounded cornea is directly correlated with the magnitude of the epithelial wound potential that arises after injury, as shown by pharmacological enhancement or disruption of this dcEF2-3. We have demonstrated that adult subependymal NPCs undergo rapid and directed cathodal migration in vitro when exposed to an externally applied dcEF. In this protocol we describe our lab's techniques for creating a simple and effective galvanotaxis assay for high-resolution, long-term observation of directed cell body translocation (migration) on a single-cell level. This assay would be suitable for investigating the mechanisms that regulate dcEF transduction into cellular motility through the use of transgenic or knockout mice, short interfering RNA, or specific receptor agonists/antagonists.

In this protocol we demonstrate how to construct custom chambers that permit the application of a direct current electric field to enable time-lapse imaging of adult brain derived neural precursor cell translocation during galvanotaxis.

一个的趋电含量为神经前体细胞迁移的动力学分析外部施加直流电场

The discovery of neural stem and progenitor  cells (collectively termed neural precursor cells) (NPCs) in the adult  mammalian brain has led to a body of ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

在体外的方法来观察派生病人和人内皮细胞前列腺循环肿瘤细胞之间的E-选择素介导的相互作用

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...