在实验室中进行的研究工作，研究基金会赠款支持 - 佛兰德（FWO授权号码G.0545.08和G.0298.11），校际景点波兰人计划（比利时科学政策;授权号P6/28和P7/13）和嵌入式在FWO支持的研究界。鼎晖是一个博士后研究基金会 - 佛拉芒（FWO），的。作者非常感谢所有现任和前任成员实验室的分子和细胞信号（鲁汶），的SP SRINIVAS博士（印第安纳大学眼科视光学院，美国），实验室博士Leybaert（根特大学）和博士Vinken（VUB）提供了有益的讨论，优化程序或参与开发工具的研究连接蛋白半管。

1。角膜内皮细胞的分离在开始之前：隔离新鲜的眼睛，从本地屠宰场，尽快去核后，眼睛的细胞。确保眼球被摘除一个很牛的最大18个月，五分钟验尸和保存在厄尔的平衡盐溶液 - 1％碘溶液在4°C运输到实验室。 <ol><li>走眼厄尔的平衡盐溶液 - 1％碘溶液，并将其放置在培养皿（100 x 20毫米）。 </li><li>眼用溶液中含有70％乙醇消毒，冲洗Earle氏平衡盐溶液含有1％的碘。 </li><li>从这个步骤开始，在无菌罩。小心地从眼睛角膜解剖，并将其放置在陪替氏培养皿（35×10毫米），含有Earle氏平衡盐溶液的，朝上的上皮细胞层。小心删除任何剩余的虹膜组织STI会附着在角膜上，如果需要的话。 </li><li>传输角膜到另一个Earle氏平衡盐溶液的培养皿中含有与血管内皮细胞层向上的Earle氏平衡盐溶液冲洗两次。 </li><li>转移的角膜内皮细胞层，一个砂漏，这是一个杯形盘朝上，并覆盖它与生长培养基。生长培养基中的Dulbecco改进的鹰的中等含有25mM的葡萄糖，10％胎牛血清，6.6％L-谷氨酰胺，2.5微克/毫升两性霉素-B和1％抗生素 - 抗真菌剂的混合物含有10,000个单位/ ml青霉素，10,000微克/毫升链霉素和25微克/毫升两性霉素B。 </li><li>吸入移液管取出培养基。 </li><li>将300μl的胰蛋白酶溶液（0.5克/升）的角膜内皮细胞层（包括胰蛋白酶的所有步骤都是使用相同的浓度）。 </li><li>将含角膜沙漏在有盖培养皿中，并把它在公司ubator 30分钟，在37℃和5％CO 2。 </li><li>轻轻刮去离角膜内皮细胞与火抛光的勾形玻璃巴斯德吸管在无菌罩。 </li><li>吸出含有内皮细胞的溶液，然后将其添加到含有4毫升培养基中的培养瓶（25cm 2的）。 </li><li>将300微升生长培养基中，在角膜和重复刮，并添加含有内皮细胞的培养瓶中的溶液。 </li><li>再次重复最后一步（1.11）。 </li><li>放置在孵化器中于37℃和5％CO 2，在生长培养基中含有的角膜内皮细胞的培养瓶。 </li><li>两天后，加入6毫升培养基。 </li><li>每隔一天刷新培养基中生长。 </li></ol> 2。细胞培养<ol><li>取出培养基，洗涤细胞两次，Earle氏平衡盐溶液，达到汇合时（内阿布T 10天隔离后）。 </li><li> 1.5毫升胰蛋白酶溶液加入到细胞中分离，并把该烧瓶中，在孵化器（37℃，5％CO 2）为3〜4分钟。 </li><li>添加12毫升生长培养基。移液器中三次进出分散细胞和细胞计数。 </li><li>种子细胞，取决于细胞的密度，并把它们在孵化器的可变部分。准备两个腔幻灯片（面积为4.2厘米2）165000细胞的细胞计数（每cm 2的细胞密度39286）。准备一个新的通道密度为每平方厘米 6250 80 cm 2的培养瓶中，并加入新鲜的培养基中至总体积为25毫升。 </li><li>刷新介质每两天。 </li><li>达到汇合的细胞层后3〜4天。使用代细胞用于实验。 </li><li>当达到汇合在瓶中的细胞层，重复步骤2.1至2.6。通道2的细胞培养可以用于或实验。 </li></ol> 3。机械刺激诱发的钙波<ol><li> 10微米荧光4加载的​​细胞腔幻灯片，AM在磷酸盐缓冲液中30分钟，在37°C，而轻轻晃动。 </li><li>取出荧光4 AM溶液，洗涤细胞5次，用磷酸盐缓冲盐水，孵育细胞，用磷酸盐缓冲盐水，并离开细胞之前，测量在室温下至少5分钟。 </li><li>氩激光并使用分束器488 HFT激发波长为488 nm，收集的荧光发射波长为530 nm，使用长波发射滤波器LP 505，设置针孔最低。使用油浸40X目标（空气，1.2 NA）。 ARL-67156在实验中，使用10倍的目标（空气，0.3 NA）。 </li><li>搜索一个领域中，细胞融合共聚焦显微镜。 </li><li>吸移管的位置，以便它是在45℃在相对于腔滑动接触的细胞膜。挑起短（约1秒）到一个单细胞的机械性刺激。机械刺激包括急性，短暂的变形细胞的短暂接触不到1％的细胞膜用玻璃微吸管（顶端直径小于1微米）耦合到一个压电晶体纳米定位，这是通过放大器操作安装在一个微操纵器。用微电极拉马玻璃微。请确定由0.2和1.5伏之间的电压的机械性刺激过程中，纳米定位操作。高于1.5 V的电压可能会导致细胞损伤。无细胞损伤的机械性刺激的最优电压的每个单元格的类型和条件，必须仔细确定通过应用一系列从低（0.2 V）高电压（1.5伏）的电压。的电压，因为这个电压决定施加到细胞膜的机械应力和应变力的刺激是衡量。该可以计算出机械性刺激的力乘以与该区域的机械应力。由于这两种区域（&lt;3.14微米2）和机械应力都非常低，低的力的机械性刺激。 （需要注意的是，当细胞被破坏，荧光漏出的细胞和细胞变暗。） </li><li>测量空间变化的[Ca 2 +] i的机械性刺激后共聚焦显微镜。 </li><li>收集和存储图像。 </li><li>绘制一个多边形区域，感兴趣的定义的共聚焦显微镜使用该软件的应答细胞（有效面积，AA）的总表面面积。 </li></ol>

作者什么都没有透露。

在这篇稿件中，我们描述了一个简单的方法来衡量初级牛角膜内皮细胞的单层细胞间的Ca 2 +波传播提供本地化和控制的机械性刺激，使用微量。机械刺激的细胞响应与局部增加细胞内的IP 3与Ca 2 +，这两者都是必不可少的细胞内信号分子，该驱动器间的Ca 2 +波传播11,67。 IP 3直接传送给通过间隙连接通道5的相邻小区，而Ca 2 +的...

间的通信和信号是必不可少的响应外兴奋剂组织和整个器官水平1,2协调的生理过程。间沟通的最直接的方法是创建发生间隙路口。缝隙连接的间隙连接通道的噬菌斑，这是蛋白质形成的通道由两个间隙连接蛋白（Cx的）相邻小区的半管3,4（ 图1）的头-头对接。缝隙连接的允许通过的小的信号分子，其分子量小于1.5 kDa的，包括的Ca 2 +或IP 3 5，引起和调制的Ca 2 +释放的相邻小区6（ 图2）从细胞内储存。间隙连接通道内和分子间的蛋白质相互作用，细胞信号传导过程，如氧化还原修改和严格的监管磷酸化7。 GJS促进细胞连接的协调反应，从而作为一种化学和电气合胞体。例如，基于Cx的GJ通道85是介导的跨心房和心室肌细胞的心肌动作电位的传播。 Cxs的不仅有间隙连接通道的作用，而且还形成未配对的半管，从而作为膜通道的常规离子通道8-10（ 图1）类似。半管参与相邻细胞之间的旁分泌信号，通过控制离子交换和信号分子之间的内和细胞外的环境。 在许多类型的细胞（如上皮细胞，成骨细胞，星形胶质细胞，血管内皮细胞等 ）和器官（如脑，肝，视网膜，耳蜗和脉管系统）中，细胞间的Ca 2 +的波的基本协调多细胞反应的<su对&gt; 11。在细胞内Ca 2 +的水平，在目标细胞中增加并不限于此细胞，而是传播到周边的相邻小区，从而建立的胞内Ca 2 +的波12,13。这些胞内Ca 2 +的波是重要的，正常的生理调节的细胞层为合胞体和它们的调节异常已与病理生理过程11。角膜内皮细胞和上皮细胞，研究不同的群体，包括我们自己的25-33，14-24间通讯的机制和作用。在非兴奋细胞，角膜内皮细胞一样，会发生两种不同的模式间的沟通28,29，即细胞间隙连接通讯和旁分泌细胞间的沟通。细胞间隙连接通讯涉及直接交换的信号分子通过间隙连接7。峡JUNC的为维持组织的动态平衡，控制细胞的增殖，并建立同步响应外应力10,34,35，周志武间的沟通非常重要。一些病症，间隙连接耦合减少因缺陷CXS，，并特此影响细胞间隙连接通讯36。这强调在多细胞生物中细胞间隙连接通讯的重要性和影响力。间隙连接通讯相比，旁分泌细胞间的通信是不依赖于细胞与细胞间的同位语，因为它涉及的释放扩散外的使者（ 图2）。不同类型的信号分子在细胞外空间的信号细胞释放的。的分子被运送到目标小区，如果检测到特定的受体蛋白质。随后受体信号复合物诱导细胞反应，被终止的信号，失活或脱敏除去。发布亲脂性细胞外信号的使者渗透膜，并作用于细胞内的受体。与此相反，亲水性信使不交叉响应细胞质膜的，但作为表面表达的受体蛋白质，然后中继信号到细胞内环境结合的配位体。三个主要家族的细胞表面受体蛋白参与这个过程：离子通道相连，酶联，G蛋白挂钩。释放的信使分子相同的细胞受体的自分泌，可作用于靶细胞上在靠近（旁分泌），或在远距离的靶细胞需要的循环系统（内分泌）。 在许多类型的细胞，包括角膜内皮细胞28,29，ATP是主要的亲水性的，该驱动器的传播间的Ca 2 +波37-40的旁分泌因子之一。期间通过各种代理的机械变形，缺氧，炎症或刺激，ATP可以释放健康的细胞41-44剪切应力，拉伸，或渗透压肿胀44,45的响应。已经假定，包括囊泡胞吐的运输机制，如ATP结合盒（ABC）转运，质膜电压依赖性阴离子通道46，P2X7受体通道47,48，以及44和大量不同的ATP释放机制 连接蛋白49-52和pannexin半管半管43,49,53。外ATP可迅速水解为ADP，AMP和腺苷54,55由ectonucleotidases是存在于细胞外的环境中。细胞释放ATP及其代谢产物ADP 56将通过扩散蔓延。随后的这些核苷酸与在相邻小区中的嘌呤能受体的相互作用已被牵连在propagation间的Ca 2 +波28,37,51。存在两个不同的类，嘌呤能受体：腺苷P1-嘌呤受体的天然配体是主要的，而嘌呤（ATP，ADP）和嘧啶核苷酸（UTP，UDP）作用于P2-嘌呤受体57。 间的通信可以通过不同的方法，如刮加载，染料转移，本地的uncaging受体激动剂如IP 3和Ca 2 +的，机械性刺激等进行调查。这里，我们描述的研究的Ca 2 +波传播引起的机械性刺激的单个单元格。研究Ca 2 +的波传播的机械刺激的优点是，它提供了一个简单的工具，以量化的Ca 2 +波的传播，随着时间的推移，它允许定量比较不同预处理细胞。在角膜内皮细胞中，这些胞内Ca 2 +的波允许的共的单层的协调响应，在此作为一个可能的防御机制帮助承受外应力，在眼内手术中的血管内皮细胞的非再生的角膜内皮细胞，或在曝光时的免疫排斥反应过程中的炎症介质或葡萄膜炎58,59。

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Column1</td>
 </tr>
 <tr>
 <td>Earle's Balanced Salt Solution (EBSS)</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>14155-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Iodine</td>
 <td>Sigma-Aldrich (Deisenhofen, Germany)</td>
 <td>38060-1EA</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>11960-044</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine (Glutamax)</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Amphotericin-B</td>
 <td>Sigma-Aldrich (Deisenhofen, Germany)</td>
 <td>A2942</td>
 <td></td>
 </tr>
 <tr>
 <td>Antibiotic-antimycotic mixture</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>15240-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>25300-054</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>14190-091</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-4 AM</td>
 <td>Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)</td>
 <td>F14217</td>
 <td></td>
 </tr>
 <tr>
 <td>ARL-67156 (6-N,N-Diethyl-b,g-dibromomethylene-D-ATP) </td>
 <td>Sigma-Aldrich (Deisenhofen, Germany)</td>
 <td>A265</td>
 <td></td>
 </tr>
 <tr>
 <td>Apyrase VI</td>
 <td>Sigma-Aldrich (Deisenhofen, Germany)</td>
 <td>A6410</td>
 <td></td>
 </tr>
 <tr>
 <td>Apyrase VII</td>
 <td>Sigma-Aldrich (Deisenhofen, Germany)</td>
 <td>A6535</td>
 <td></td>
 </tr>
 <tr>
 <td>Gap26 (VCYDKSFPISHVR)</td>
 <td>Custom peptide synthesis</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gap27 (SRPTEKTIFII)</td>
 <td>Custom peptide synthesis</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Control Peptide (SRGGEKNVFIV)</td>
 <td>Custom peptide synthesis</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>siRNA1 Cx43 (sense: 5'GAAGGAGGAGGAACU-CAAAdTdT) </td>
 <td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>siRNA2 Cx43 (sense: 5'CAAUUCUUCCUGCCGCAAUdTdT) </td>
 <td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>siRNA scramble: scrambled sequence of siCx43-1 (sense: 5'GGUAAACG-GAACGAGAAGAdTdT) </td>
 <td>Annealed siRNA was purchased at Eurogentec (Luik, Belgium)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TAT-L2 (TAT- DGANVDMHLKQIEIKKFKYGIEEHGK)</td>
 <td>Thermo Electron (Ulm, Germany)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TAT-L2-H126K/I130N (TAT-DGANVDMKLKQNEIKKFKYGIEEHGK) </td>
 <td>Thermo Electron (Ulm, Germany)</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Two chambered glass slides</td>
 <td>Laboratory-Tek Nunc (Roskilde, Denmark)</td>
 <td>155380</td>
 <td></td>
 </tr>
 <tr>
 <td>Confocal microscope </td>
 <td>Carl Zeiss Meditec (Jena, Germany)</td>
 <td>LSM510</td>
 <td></td>
 </tr>
 <tr>
 <td>Piezoelectric crystal nanopositioner (Piezo Flexure NanoPositioner)</td>
 <td>PI Polytech (Karlsruhe, Germany)</td>
 <td>P-280</td>
 <td></td>
 </tr>
 <tr>
 <td>HVPZT-amplifier</td>
 <td>PI Polytech (Karlsruhe, Germany)</td>
 <td>E463 HVPZT-amplifier</td>
 <td></td>
 </tr>
 <tr>
 <td>Glass tubes (glass replacement 3.5 nanoliter)</td>
 <td>World Precision Instruments, Inc. Sarasota, Florida, USA</td>
 <td>4878</td>
 <td></td>
 </tr>
 <tr>
 <td>Microelectrode puller </td>
 <td>Zeitz Instrumente (Munchen, Germany)</td>
 <td>WZ DMZ-Universal Puller</td>
 <td></td>
 </tr>
</tbody>
 </table>

mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells

间的沟通是必不可少的各种器官和组织，包括大脑，肝脏，视网膜，耳蜗血管细胞之间的生理过程的协调。在实验设置中，细胞间的Ca 2 +波可以引起的施加机械刺激的单个单元格。这会导致细胞内信号分子IP 3与Ca 2 +的释放，启 ​​动同心的机械刺激细胞的Ca 2 +波传播到邻近细胞。的主要分子间的Ca 2 +波传播途径，控制是通过直接传输IP 3和半管通过ATP释放的间隙连接通道。鉴定和表征的属性和调节不同连接蛋白和pannexin的亚型，间隙连接通道和半管所允许的quantification的胞间的Ca 2 +波，小干扰RNA，和间隙连接通道和半管抑制剂的使用的蔓延。在这里，我们描述的方法来衡量间的Ca 2 +波原发性角膜内皮细胞的单层Fluo4-AM加载响应的控制和局部机械的刺激引起的一种急性，短暂变形的细胞，结果接触，用显微操纵器控制与齿顶圆直径小于1微米的玻璃微细胞膜。我们还描述了初级牛角膜内皮细胞和其用作模型系统评估Cx43的hemichannel活性间的Ca 2 +波通过释放的ATP作为驱动力的隔离。最后，我们讨论的使用，优点，局限性和替代品的差距结的通道和hemichannel研究的背景下，这种方法。

执行所有的实验都符合所有有关准则，法规和监管机构的协议被证明是鲁汶的动物护理和使用委员会的指导和批准下进行。 牛角膜内皮细胞（BCEC），表达和功能的间隙连接两种细胞间隙连接通讯和旁分泌细胞间通信间的沟通互动的方式作出显著贡献，但已被证明的主要途径是旁分泌细胞间的通信通路介导的通过ATP释放Cx43的半管28,29。 ATP和ADP是由ectonucleotidase电子NTPD1?...

Watch this Scientific Journal Video about Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells at JoVE.com

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

细胞间钙<sup&gt; +</sup&gt;波驱动的间隙连接通道和半管。在这里，我们描述了一种方法来测量细胞间钙<sup&gt; +</sup&gt;波在细胞模型在响应到本地单细胞的机械刺激和调查中的应用性能和调节间隙连接通道和半管。

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

mechanical-stimulation-induced-calcium-wave-propagation-cell

Research

JoVE Journal

Biology

16.1K 次观看.  KU Leuven. 细胞间钙 +波驱动的间隙连接通道和半管。在这里，我们描述了一种方法来测量细胞间钙 +波在细胞模型在响应到本地单细胞的机械刺激和调查中的应用性能和调节间隙连接通道和半管。

视频: Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

表达GFP -肌动蛋白的内皮细胞的肌动蛋白细胞骨架的研究

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...