作者承认资金从澳大利亚研究理事会和国家健康与医学研究委员会澳大利亚也想感谢 澳大利亚国家的制造工厂主的微接触印刷的SU-8。 SHN支持的马来西亚高等教育和马来西亚理科大学部。

1。 PLL-PEG-生物素玻片上与钝化这一步的目的是钝化的表面，从而使细胞的粘附和迁移到特定区域的盖玻片上所建立的（步骤2-3A）microcontract印刷或的蘸水笔平版印刷（步骤3b）。对于钝化，一个共同的聚合物的聚-L-赖氨酸（PLL）和聚乙二醇（PEG）中，使用20％的PEG分子被接枝到生物素（PLL-PEG-生物素）。 <ol><li>要清洁的玻璃盖片（18毫米×18毫米），浸没在100％乙醇中的齿条，和放置在机架中的声波处理30分钟，浴。擦干玻璃盖玻片，在空气或氮气流中下。 </li><li>接着，在1 M NaOH中的30分钟超声的玻璃盖玻片。事后冲洗玻璃表面通过仔细的机架中浸入烧杯中充满了1.5 L的Milli-Q H 2 O新鲜的Milli-Q H 2 O每次使用三次重复冲洗过程。干燥盖玻片在60℃的烘箱</li><li>将一半的干净的玻璃盖玻片单独在潮湿的腔室部分地填充有H 2 O 24 -孔的板，它是由掉落PLL-PEG-生物素（1毫克/毫升），在PBS中的15μl的每个玻璃盖玻片上。以剩余的干净的玻璃盖玻片的另一半和仔细夹心的PEG溶液。将盖玻片，在潮湿室1小时。轻轻滑动夹持盖玻片远离彼此不刮的PEG-涂覆的表面的情况下，他们用Milli-Q H 2 O冲洗水应该很容易滑落的PEG处理侧。如果需要，空气干燥的玻璃盖玻片纸巾上与处理过的表面朝上。存储的玻璃盖玻片，在真空干燥器中，直到进一步使用。 </li></ol> 2。微接触印刷邮票的制作我们描述了两种模式的链霉亲和轨道的过程钝化的盖玻片上。第一个，话筒rocontact印刷，通常需要制作的PDMS印章硅主投。在我们的例子中，PDMS印章上的图案是为100μm宽的线的特征高度为25μm，间距为290μm的中心到中心的。下面，我们简单地描述了如何制作一个合适的硅主（2.1-2.3）和PDMS印章（2.4）。但是，也有文献19-23，这是适合用于打印到盖玻片上的蛋白质模式中描述的微接触印刷的多个协议。大师也可以进行定制，并从商业渠道购买。 <ol><li>首先，在硅晶片（4"直径）清洁Piranha溶液（3:1体积比的硫酸和30％的过氧化氢 ​​）中孵育20分钟。彻底冲洗晶片用MilliQ H 2 O中的 10倍干燥的晶片在烘箱中在150℃CO / N。 </li><li>冲洗干净并干燥的硅晶片由100％的异丙醇，然后用100％丙酮。下旋涂2毫升GM1070 SU-8的光刻胶（Gersteltec Sarl的）在3100转的晶片上，持续40秒，以创建一个25μm厚的层。然后将样品在65℃的热板上烘烤5分钟，然后通过进一步的5分钟，在95℃下</li><li>在SU-8的光致抗蚀剂暴露于UV光下，进行60秒的所需图案的光掩模（例如，使用掩模对准器Quintel Q6000），并重复烘烤过程中在2.2中描述。然后样品被开发的晶片浸渍在SU-8显影剂（Gersteltec Sarl公司）为4分钟，在100％异丙醇冲洗并在氮气流中干燥。由此除去未暴露于UV光的SU-8的光致抗蚀剂的区域的，创建的逆图案的PDMS印章。 </li><li>下一步是从硅主投PDMS印章。首先，彻底混合1份（重量/重量）的固化剂的弹性体预聚物与10份（Sylgard的184）。中去除任何空气气泡，脱气混合物在干燥器中连接到真空管线为1小时。接下来，倒入到陪替氏培养皿内的硅主的PDMS弹性体至约1厘米的高度，并在烘箱中在70℃下固化该主站和PDMS混合物1小时。最后，除去PDMS印章从硅主站，并切割成一定的尺寸。 </li></ol> 3。钝化的盖玻片上的图案链霉亲和素或粘合剂线索钝化盖玻片后，蛋白质模式上都印有微接触印刷（步骤3a）或蘸笔光刻技术（步骤3b）。在我们的例子中，链霉抗生物素蛋白的打印线，RGD梯度（步骤4）形成的基础。相同的步骤也可以用于印刷基质蛋白创建粘合剂图案。 到表面上的图案化的生物分子的微接触印刷的另一种方法是蘸笔光刻。在蘸笔光刻，以悬臂是用来传输一个小的表面上，溶液的体积。悬臂，V的大小iscosity的解决方案，在悬臂的表面上的接触时间，表面的疏水性和湿度在印刷室确定的大小印刷的功能。此方法的优点是，不同的图案可印每个实验，因为没有必要制作主人和邮票。它的缺点是，它需要更长的时间来打印一个给定模式和，蘸笔光刻技术需要专门的仪器可能无法使用在许多实验室。蘸水笔光刻工具，我们使用的是从Bioforce纳米NanoeNabler。在这里，我们印小点直径&lt;10微米，我们SPT10悬臂。 3A。 "微接触的印刷（μCP）技术<ol><li>要清洁和使PDMS印章更具亲水性，在1.5小时的紫外线和臭氧（例如，紫外线臭氧Procleaner奈米从Bioforce）的PDMS邮票治疗图案侧。或者，也可以使用等离子吸尘器用于相同目的的时间更短。此过程创建的氧化的PDMS表面24，从而提高了印刷过程。 </li><li>后立即臭氧或等离子处理，将10微升的链霉亲和素（1毫克/毫升在PBS中）和传播到PDMS邮票和离开1小时，在潮湿的腔室（例如在部分充满的6孔板中，见步骤1.3） 。如果轨道应该是在荧光显微镜下可见，使用链霉抗生物素蛋白，如链霉抗生物素蛋白-AlexaFluor350荧光团共轭。 </li><li>删除多余的链霉亲和素的邮票用纸巾和空气干燥的邮票约1分钟。 PLL-PEG-生物素包被盖玻片上，轻轻按下邮票。 </li><li>如果荧光链霉亲和素使用在步骤3.a2，检查落射荧光显微镜下使用的模式。印刷面储存在干燥器中。 </li></ol> 3b所示。浸笔微影技术<ol><li>首先，将1份为1毫克/毫升链霉亲和素或STREptavidin-AlexaFluor350甘油与10份（体积/体积），并加载到悬臂5微升该混合物。然后开始印刷过程中，，蘸水笔光刻仪器的操作手册中所描述的。为了减少到约5μm的光斑尺寸，调整印刷速度，接触时间在悬臂的悬臂的表面上的表面或垂直距离。在图2c中所示的例子中，印刷在设置为10-15μm的行和列的分离的线与点。这个距离是足够的，以防止点从彼此合并成，但允许在一个字段中的观点与大多数显微镜设置多个点的观看。 </li><li>印刷面储存在干燥器中。 </li></ol> 4。创建链霉亲和型面的胶渐变到我们创建粘合剂梯度生物素-RGD的玻璃盖玻片上都涂有链霉抗生物素蛋白（ 图3）或共聚ntain钝化玻璃盖玻片上的链霉抗生物素蛋白的模式（ 图5）。要创建一个表面的倾斜度，我们使用市售微流体装置（称为粘性滑动趋化三维 Ibidi）的梯度通过被动扩散，创建一个稳定的解决方案。为链霉抗生物素蛋白的结合位点的竞争，我们采用两个相对的梯度是不共轭的RGD序列（参见图1）的生物素-RGD和生物素。 <ol><li>坚持的链霉亲和素包被或图案的盖玻片上的粘性面的粘幻灯片趋化3D设备。为了确保有几个小时是无泄漏，而装置边缘有一薄层的温热凡士林和石蜡的1份（重量/重量）的混合物1份凡士林用第二层密封。 </li><li>填充与6μlPBS中的信道的信道的设备。然后填写70微升0.03μg/μL的生物素 - 荧光素在PBS和70米的两个水库U;升0.03微克/μL生物素-RGD的PBS，分别。的样品在室温下孵育1小时，在黑暗。 </li><li>移除的生物素溶液，并同时仍连接到的微流体装置，两次用PBS仔细冲洗表面。马克的粘接剂梯度的方向，并且如果需要的话，储存在4℃下用PBS填充设备，但确保该设备不干燥。如果需要，粘合剂梯度用荧光显微镜进行分析。 </li></ol> 5。标签的萤光细胞与活细胞成像将细胞用荧光染料，协助细胞在荧光显微镜下的跟踪标记。常见的探针荧光的细胞器标记物，如祥发64红，标记细胞核。要做到这一点，细胞首先被洗涤三次，用PBS 1μM祥发64红45分钟的培养基中，然后孵育，最后用PBS洗涤进一步三次。请注意，细胞不应该是在PBS中保持很长一段时间。 替代染料的细胞追踪CellTracker系列染色的细胞质，并保留在细胞迁移和分裂。转染的细胞与荧光融合蛋白是特别有用的，在迁移过程中记录感兴趣的蛋白的亚细胞分布。然而，典型的光漂白剂的速度比有机染料的荧光蛋白，使长期观测不可以有可能。 6。载入细胞的渐变成一种可溶性的微流体装置<ol><li>计数荧光标记的细胞分裂成两管相同的手机号码。洗净，重悬于一个管与媒体含有低葡萄糖Dulbecco的修改的Eagle培养基（DMEM）中，用10％胎牛血清（FBS）的趋化因子，如细胞。洗净，在媒体的细胞没有趋化， 即 0％FBS的DMEM悬浮管。的最终浓度每个试管中的细胞应为5×10 5细胞/ ml。 </li><li>删除所有的解决方案，微流体装置（4.3），并把它放在显微镜载物台（见第7步）。 70微升的细胞（5×10 5个细胞/毫升0％FBS的DMEM中）加载到一个水库和70微升的HeLa细胞（5×10 5个细胞/毫升在10％FBS的DMEM中）到另一个。松松地将磁带的水库，以避免蒸发。如果细胞的表面上的预孵育成像之前，可取的做法是将细胞生长介质中的装置装入未经梯度。该装置然后被放置在显微镜载物台上，并用不含FBS的DMEM培养液梯度介质， 即在一个贮存器和DMEM含10％FBS在其他媒体取代。 </li></ol> 7。活细胞成像和数据分析<ol><li>开关上的显微镜（尼康的Ti-E）和预热阶段在实验开始前至少1小时。 </li><li>确保通道的MICRofluidic的移动设备的视场中，细胞是在焦点。选择图像采集参数，如曝光时间和荧光过滤器，以及明场和荧光捕获细胞和曲目的图像序列的时间点，记录长度（ 例如，每15分钟16小时）。 </li><li>分析数据与适当的软件，如ImageJ的手动跟踪或者Ibidi的趋化和迁移工具。 </li></ol>

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H., Jiang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Cell+Migration+on+Surface-Density+Gradients+of+Fibronectin,+VEGF,+or+Both+Proteins.">Endothelial Cell Migration on Surface-Density Gradients of Fibronectin, VEGF, or Both Proteins.</a> Langmuir. 23, 11168-11173 (2007).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+cell+migration:+a+multiscale+tuning+model.">Plasticity of cell migration: a multiscale tuning model.</a> The Journal of Cell Biology. 188, 11-19 (2010).</li><li>Moazzam, F., DeLano, F. A., Zweifach, B. W., Schmid-Sch&#246;nbein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+leukocyte+response+to+fluid.">The leukocyte response to fluid.</a> Proceedings of the National Academy of Sciences. 94, 5338-5343 (1997).</li><li>Walker, G. M., 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=Effects+of+flow+and+diffusion+on+chemotaxis+studies+in+a+microfabricated+gradient+generator.">Effects of flow and diffusion on chemotaxis studies in a microfabricated gradient generator.</a> Lab on a Chip. 5, 611-618 (2005).</li><li>Perl, A., Reinhoudt, D. N., Huskens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing:+Limitations+and+Achievements.">Microcontact Printing: Limitations and Achievements.</a> Adv. Mater. 21, 2257-2268 (2009).</li><li>Salaita, K., Wang, Y., Mirkin, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+dip-pen+nanolithography.">Applications of dip-pen nanolithography.</a> Nat. Nano. 2, 145-155 (2007).</li><li>Wouters, D., Schubert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolithography+and+nanochemistry:+probe-related+patterning+techniques+and+chemical+modification+for+nanometer-sized+devices.">Nanolithography and nanochemistry: probe-related patterning techniques and chemical modification for nanometer-sized devices.</a> Angewandte Chemie (International ed. in English). 43, 2480-2495 (2004).</li><li>Xia, N., 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=Directional+control+of+cell+motility+through+focal+adhesion+positioning+and+spatial+control+of+Rac+activation.">Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation.</a> FASEB J. 22, 1649-1659 (2008).</li><li>Oakes, P. W., Beckham, Y., Stricker, J., Gardel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+is+required+but+not+sufficient+for+focal+adhesion+maturation+without+a+stress+fiber+template.">Tension is required but not sufficient for focal adhesion maturation without a stress fiber template.</a> The Journal of Cell Biology. 196, 363-374 (2012).</li></ol>

我们有没有商业利益的披露。

在这个协议中，我们使用市售的微流体装置（粘性滑动的趋化三维 Ibidi）的化学表面改性及对细胞迁移的趋化因子梯度的影响进行研究。因为梯度设立的沿该通道的长度的扩散，这种微流体设置不需要流。这是很重要的，因为流量差异影响慢慢迁移的细胞，如成纤维细胞，形成稳定的粘着斑和快速迁移细胞最喜欢的白细胞，形成协调配合物与突触小26。从层流剪应力可能影响稳定?...

导演细胞迁移是许多细胞的基本属性，是许多正常的生理过程，包括胚胎发育，抗感染，促进伤口愈合的一个重要方面。此外，细胞迁移也起到了突出的作用，在许多疾病，如血管疾病，肿瘤细胞的转移和慢性炎症1,2。虽然传统的状态的细胞迁移-极化，凸出延伸，形成粘连，力产生收缩和后收缩-被普遍接受的3,4，阐明的时空信号集成协调机制，已是更具挑战性。 的胞外基质（ECM）作为细胞粘附的基板，并通过固有的化学和物理6多样性，提供用于细胞导航7,8的方向提示。除了 ​​这些粘合剂线索9，可溶性因子10-12 &lt;/ SUP&gt;，如趋化因子和生长因子，可诱导细胞迁移通过定向趋化因子受体和其下游motogenic的信令。目前，参与和信令通过粘附受体（ 即整合），或如趋化因子受体，受体酪氨酸激酶（RTK），不知道是否是显性的。也不知道是否是细胞类型特异性受体系统的层次结构。 活细胞显微镜提供了丰富的信息，是不是可以批量检测，可以结合微流体装置，产生梯度的固定13,14和可溶性线索15 16。这里描述的方法采用了一系列的简单和既定一并用市售的微流体腔室，以创建一个成像检测细胞的迁移，可以很容易地在细胞生物学实验室（ 图1）实施表面改性步骤。组装的微流体装置具有与玻璃17的光学品质的梯度的扩散的分子是稳定的，至少16小时。该系统可用于落射荧光和先进的显微镜技术。不像其他的趋化性的设置18，这个系统是适合用于记录缓慢迁移，贴壁细胞。更重要的是，该系统是模块化的，很容易使引进替代的粘合剂或可溶性迁移暗示的各种细胞类型和检查。

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Coverglass staining outfits</td>
 <td>Thomas Scientific</td>
 <td>8542 E40</td>
 <td>Coverslip rack</td>
 </tr>
 <tr>
 <td>Oven</td>
 <td>Binder</td>
 <td></td>
 <td>ED 53 series</td>
 </tr>
 <tr>
 <td>Silicon wafer</td>
 <td>Silicon Quest</td>
 <td>708-007</td>
 <td>Boron doped &lt;100&gt; wafer, 4" diameter, 500 &mu;m, single side polished</td>
 </tr>
 <tr>
 <td>GM1070 SU-8 photoresist</td>
 <td>Gersteltec Sarl</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SU-8 developer</td>
 <td>Gersteltec Sarl</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 curing agent</td>
 <td>Dow Corning</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 elastomer prepolymer</td>
 <td>Dow Corning</td>
 <td></td>
 <td >&nbsp;</td>
 </tr>
 <tr>
 <td>PLL-PEG-biotin (20%)</td>
 <td>SuSos AG</td>
 <td>PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)</td>
 <td>1 mg/ml in PBS</td>
 </tr>
 <tr>
 <td>Fluorescein</td>
 <td>Sigma</td>
 <td>46955</td>
 <td>1 mM in PBS</td>
 </tr>
 <tr>
 <td>Streptavidin-AlexaFluor350</td>
 <td>Invitrogen</td>
 <td>S-11249</td>
 <td>1 mg/ml in PBS</td>
 </tr>
 <tr>
 <td>Biotin-4-fluorescein</td>
 <td>Invitrogen</td>
 <td>B-1370</td>
 <td>0.03 &mu;g/&mu;l in PBS</td>
 </tr>
 <tr>
 <td>Biotin-RGD</td>
 <td>GenScript</td>
 <td>SC1208</td>
 <td>0.03 mg/ml in PBS</td>
 </tr>
 <tr>
 <td>Syto 64 Red</td>
 <td>Invitrogen</td>
 <td> S-11346 </td>
 <td>1 &mu;M in PBS</td>
 </tr>
 <tr>
 <td>Sticky slide chemotaxis 3D</td>
 <td>Ibidi</td>
 <td>80328</td>
 <td></td>
 </tr>
 <tr>
 <td>200 &mu;l Greiner yellow bevelled tip</td>
 <td>Greiner Bio-One</td>
 <td>739261</td>
 <td></td>
 </tr>
 <tr>
 <td>Vaseline</td>
 <td>Sigma</td>
 <td>16415</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraffin wax, mp 55-57 &deg;C</td>
 <td>Sigma</td>
 <td>327204</td>
 <td></td>
 </tr>
 <tr>
 <td>Nano eNabler 10 &mu;m cantilever </td>
 <td>BioForce</td>
 <td>SPT-S-C-10s</td>
 <td></td>
 </tr>
 <tr>
 <td>Image J software</td>
 <td>National Institute of Health</td>
 <td></td>
 <td ><a href="http://rsbweb.nih.gov/ij/download.html" target="_blank">rsbweb.nih.gov/ij/download.html</a></td>
 </tr>
 <tr>
 <td>Manual Tracking plugin</td>
 <td>Fabrice Cordeli&egrave;res</td>
 <td></td>
 <td ><a href="http://rsb.info.nih.gov/ij/plugins/track/track.html" target="_blank">rsb.info.nih.gov/ij/plugins/track/track.html</a></td>
 </tr>
 <tr>
 <td>Chemotaxis and Migration Tool</td>
 <td>Ibidi GmbH</td>
 <td></td>
 <td><a href="http://www.ibidi.com/applications/ap_chemotaxis.html" target="_blank">www.ibidi.com/applications/ap_chemotaxis.html</a></td>
 </tr>
	 </tbody>
 </table>

creating adhesive and soluble gradients for imaging cell migration with fluorescence microscopy

细胞可以感知并移向较高浓度的粘合剂线索，如糖蛋白如生长因子的细胞外基质和可溶性线索。在这里，我们列出了反对梯度的粘合剂和可溶性线索，微流控室，这是兼容与活细胞成像的方法来创建。聚-L-赖氨酸和聚乙二醇（PLL-PEG）的共聚物，以钝化玻璃的盖玻片和防止生物分子和细胞的非特异性吸附。往下，微接触印刷或蘸笔光刻被用来创建轨道的链霉抗生物素蛋白上的钝化表面的生物素化的肽，精氨酸 - 甘氨酸 - 天门冬氨酸（RGD）作为粘接剂提示作为锚固点。一种微流体装置被放置到改性的表面上，并用于创建的梯度粘合剂线索（100％到0％的RGD RGD）上的抗生蛋白链菌素的轨道。最后，相同的微流体装置是用于创建的单核细胞趋化自吸的梯度h为牛胎儿血清（FBS），在相反的方向上的梯度的粘合剂线索作为可溶性提示。

为了了解细胞整合洄游信号25，我们已经开发了一种方法的图像细胞用荧光显微镜观察，与竞争的粘接剂和可溶性的梯度（ 图1）的环境中迁移。胶粘剂的轨道含有荧光链霉亲和素和生物素标记的RGD创建的微接触印刷的曲目和蘸笔光刻（ 图2）。成功的微接触印刷粘合剂轨道和防污区域可以清楚地加以区分（ 图2b）的磁道之间的荧光强度表示的线剖面?...

Watch this Scientific Journal Video about Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy at JoVE.com

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

为组件的粘接剂和可溶性的梯度在显微镜室，用于活细胞迁移研究的方法，进行说明。该工程环境结合溶液梯度的防污表面和粘接剂的轨道，并因此允许一个确定指导线索的相对重要性。

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

creating-adhesive-soluble-gradients-for-imaging-cell-migration-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.5K 次观看.  The University of New South Wales. 为组件的粘接剂和可溶性的梯度在显微镜室，用于活细胞迁移研究的方法，进行说明。该工程环境结合溶液梯度的防污表面和粘接剂的轨道，并因此允许一个确定指导线索的相对重要性。

视频: Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

柔性神经微探针的使用附加与Biodissolvable胶粘剂刚性加劲肋插入

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...