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?...

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 border="0" cellpadding="0" cellspacing="0" width="774">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cells</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pRNS-1-1 prostate cells</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td>Lee et. al., (1994)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PNT2 prostate cells</td>
			<td style="width:117px;">Sigma-Aldrich</td>
			<td style="width:119px;">95012613-1VL</td>
			<td>Berthon et al., (1995)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium and solutions</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 medium&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">11875-093</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum - Premium</td>
			<td style="width:117px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>10% in PBS, warm up to 37&#176;C before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Antibiotic-Antimycotic (100x)</td>
			<td style="width:117px;">Life Technologies</td>
			<td style="width:119px;">15240</td>
			<td>add 5mL to 500mL medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-propanol</td>
			<td style="width:117px;">VWR</td>
			<td>BDH1133-5GL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:117px;">-</td>
			<td style="width:119px;">-</td>
			<td>137mM NaCl, 2.7mM KCl, 4.3mM Na2HPO4 and 1.5mM KH2PO4 in 1000mL of H2O, pH to 7.4 and autoclaved, warm up to 37&#176;C before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA&#160;</td>
			<td style="width:117px;">Invitrogen</td>
			<td style="width:119px;">25200-056</td>
			<td>warm up to 37&#176;C before use, treat cells for 3-5 min at 37&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galvanotaxis device</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis chambers</td>
			<td style="width:117px;">Precision Plastics Inc, CA</td>
			<td style="width:119px;"></td>
			<td>custom-designed (1/4&#34; X 2&#34; X 3.5&#34;), non-toxic, clear PVC chambers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis electrodes</td>
			<td style="width:117px;">UCD electric shop</td>
			<td style="width:119px;"></td>
			<td>platinum coiled electrodes with flexable cords</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Galvanotaxis power box</td>
			<td style="width:117px;">Substrate Engineering, CA</td>
			<td style="width:119px;"></td>
			<td>custom-designed DC power output with voltmeter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope Cover Glass, Large, 45X50mm-No. 1.5</td>
			<td style="width:117px;">Fisher</td>
			<td style="width:119px;">12-544-F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope Cover Glass, small, 25X25mm, No. 1</td>
			<td style="width:117px;">ThermoScientific</td>
			<td style="width:119px;">3307</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond point marker</td>
			<td style="width:117px;">ThermoScientific</td>
			<td style="width:119px;">750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Marine grade silicon sealer, clear</td>
			<td style="width:117px;">3M</td>
			<td style="width:119px;">051135-08019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease</td>
			<td style="width:117px;">Dow Corning</td>
			<td style="width:119px;">2021846-0807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6mL syringe</td>
			<td style="width:117px;">Fisher Scientific</td>
			<td style="width:119px;">05-561-64</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nichiryo Syringe, 1.5mL</td>
			<td style="width:117px;">Nichiryo</td>
			<td style="width:119px;">SG-M</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cotton applicators</td>
			<td style="width:117px;">Purtian Medical Products</td>
			<td style="width:119px;">806-WC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Qtips</td>
			<td style="width:117px;">Johnson &#38; Johnson</td>
			<td style="width:119px;">729389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene 180 PVC tubing&#160;</td>
			<td style="width:117px;">Nalgene</td>
			<td style="width:119px;">8000-9030</td>
			<td>503/16 ID x 5/16 OD x 1/16 Wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-Agar</td>
			<td style="width:117px;">Difco</td>
			<td style="width:119px;">0140-01</td>
			<td>make 2% agar solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Razor Blade</td>
			<td style="width:117px;">Personna</td>
			<td style="width:119px;">74-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipments and Software</td>
			<td style="width:117px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Benchtop Centrifuge</td>
			<td style="width:117px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td>operated with an A-4-62 rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellometer Auto T4</td>
			<td style="width:117px;">Nexcelom</td>
			<td style="width:119px;">Auto T4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellometer counting chambers</td>
			<td style="width:117px;">Nexcelom</td>
			<td style="width:119px;">CHT4-SD100-002</td>
			<td>load 20uL cell solutions to count</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Culture Temp Warming plate</td>
			<td style="width:117px;">Bel-Art Scienceware</td>
			<td style="width:119px;">370150000</td>
			<td>to keep the galvanotaxis chambers at 37&#176;C&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Eclipse TE-2000 microscope with motorized stage and environmental chamber</td>
			<td style="width:117px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plan Fluor 10X/0.30 objective len</td>
			<td style="width:117px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retiga EX CCD camera&#160;</td>
			<td style="width:117px;">Qimaging</td>
			<td style="width:119px;"></td>
			<td>Cooled CCD camara, mono-color, 12-bit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compressed air with 5% CO2&#160;</td>
			<td style="width:117px;">Airgas</td>
			<td style="width:119px;"></td>
			<td>special order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volocity 6.3</td>
			<td style="width:117px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>Image acquiring software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Improvision OpenLab 5.5.2</td>
			<td style="width:117px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td>Cell tracking software and customized to measure migration angles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FileMaker Pro Advanced, 8.0</td>
			<td style="width:117px;">FileMaker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microsoft Excel 2008 for Mac</td>
			<td style="width:117px;">Microsoft</td>
			<td style="width:119px;"></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。 <p class="jove_c...

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

计算机生成的动物模型的刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

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 ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

采用Caco-2细胞研究脂质运输由肠

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.