ML支持，由NIA（R00AG033176和R01AG040081）和实验室指导研究和发展，美国能源部能源合同＃DE-AC02-05CH11231。

1。印刷基质准备使用聚二甲基硅氧烷（PDMS）涂层或聚丙烯酰胺（PA）涂层幻灯片的决定取决于实验设计的重要参数。两种聚合物的弹性模量能够被调谐到不同的组织中，通过改变碱/固化的PDMS比，和丙烯酰胺/双丙烯酰胺的比例的PA的刚度模仿。 PDMS可以模仿较硬组织在1-10MPa的（ 如软骨，角膜，动脉壁）的范围内，和PA可以模仿较软组织中的范围为100Pa-100kPa的（ 例如，乳腺癌，脑，肝，和前列腺癌）5。 PDMS是价格便宜，易于制备，和印刷的特征的几何形状将是相同的印刷标签的头部。这样的特征的尺寸和形状，可以精确地控制，使用具有不同尖端的几何形状的标签。 PDMS是比PA更疏水性的，这会导致在细胞处理和免疫抑制的一些挑战含的步骤操作，并可能与某些细胞系中不兼容。因为PA是一种水凝胶和本地的非防污表面，细胞就会只重视有支持细胞粘附的蛋白，产生斑点。印刷的特征的几何形状，在PA凝胶不严格按照针头的几何形状，通常它们成为圈由于扩散，不论所使用的针头的几何。印刷接触时间和针直径参数可以凭经验确定了最佳的PA凝胶特征尺寸。 聚二甲基硅氧烷（PDMS） <ol><li>在一次性塑料杯，结合Sylgard的184有机硅弹性体的基础与固化剂的比例为10:1，大力木制或塑料压舌板当时钟为30分钟，在室温下真空脱气在混合。 </li><li>中心旋涂机上的真空致动夹头，一个标准的显微镜载玻片，然后毛毛雨0.5毫升到的滑动面的中心的混合弹性体聚合物。旋转在6000转60秒。 </li><li>固化的PDMS-包被的载玻片在70℃的烘箱中或上的数字的热板（保护免受灰尘）为4小时至过夜。 </li><li>固化后的幻灯片可以立即使用，或在幻灯片框，密封在一个塑料拉链袋，并保持在一个抽屉里储存数月。 PDMS吸附灰尘，所以它必须得到很好的保护，从房间空气流通。 </li><li> 注：丁腈橡胶或其他非乳胶手套时，必须佩戴工作的PDMS弹性体套件。偶然接触乳胶手套会抑制PDMS聚合。 </li></ol>聚丙烯酰胺（PA） <ol start="6"><li>的NaOH蚀刻：将上滑动的加热块在80℃下每张幻灯片上添加1毫升0.1N氢氧化钠，一定要覆盖整个滑动面。让该NaOH蒸发（应的滑动表面上形成的白色膜）。由于PA的凝胶只可以连接坚决NaOH溶液蚀刻表面上，PA凝胶在干燥过程中的整个幻灯片冲浪的步骤，如果分离ACE是不包括用NaOH。如果不完全覆盖的滑动面，重复加入1毫升0.1N的NaOH。数天，然后可被储存在室温（RT）相片数。注：NaOH溶液腐蚀的另一种方法是臭氧或等离子清洗的幻灯片。 </li><li> 3 - 氨基丙基三乙氧基硅烷（APES）涂层：在通风橱中，在15毫升菜的地方幻灯片，并添加300微升APES每个NaOH溶液蚀刻幻灯片。让的的APES反应的NaOH 5分钟滑动。超过这个时间，会造成难以洗出未反应的APES试剂。用去离子水充分洗净，满分APES在幻灯片的两侧上的两到三倍。如果清洗是不完整的，APES将氧化戊二醛形成棕色沉积在在步骤1.8的幻灯片。 </li><li>戊二醛氧化：吸滑动面的所有解决方案。在每个15 cm培养皿中，加入25毫升0.5％的戊二醛PBS。反应30分钟，在黑暗的地方。 30分钟后，吸出所有的戊二醛和使用非皮棉劳动atory湿巾（ 如 Kimwipes）的，仔细干的幻灯片。然后可被储存在RT相片数至多一天。 </li><li>凝胶制备：制备PA包括丙烯酰胺，双丙烯酰胺-丙烯酰胺和DDH 2 O的混合物中按照以下的表，脱气30分钟，然后在冰上放置PA混合物减慢聚合后。加入APS和TEMED，混合前的凝胶。移液管PA的混合物上的滑动表面和地点24毫米×50毫米，数字1的PA上的盖玻片。请避免按压盖玻片和载玻片在一起，避免泡沫的形成。对于凝胶&gt; 40,000霸用100微升，其他的凝胶用350微升。 </li></ol><table border="1"><tbody><tr><td>所需的弹性模量（Pa）的</td><td>丙烯酰胺％ </td><td>双丙烯酰胺％ </td><td>丙烯酰胺40％的股份（毫升） </td><td>双丙烯酰胺2％的股份（毫升） </td><td>去离子水（毫升） </td><td> APS（微升） </td><td> TEMED（μl） </td></tr><tr><td> 480±160 </tD&gt; <td> 3 </td><td> 0.06 </td><td> 0.75 </td><td> 0.3 </td><td> 8.95 </td><td> 100 </td><td> 10 </td></tr><tr><td> 4470±1190 </td><td> 5 </td><td> 0.15 </td><td> 1.25 </td><td> 0.75 </td><td> 8 </td><td> 100 </td><td> 10 </td></tr><tr><td> 40400±2390 </td><td> 8 </td><td> 0.48 </td><td> 2 </td><td> 2.4 </td><td> 5.6 </td><td> 100 </td><td> 10 </td></tr></tbody></table>改编自6,7。 <ol start="10"><li>让的PA凝胶聚合2小时，然后取出盖玻片下用去离子水。 </li><li>用PA在的大Coplan罐，在水中过夜（约8小时），除去未反应的丙烯酰胺凝胶幻灯片。 </li><li>在37°C的烘箱中干片为2-4小时，或直至PA凝胶完全变硬。 </li><li> PA凝胶幻灯片可以被存储在4℃下，在一个密封的滑动箱为一个月。 </li></ol> 2。蛋白质主控板准备<ol><li>所有的蛋白质shoulD是等分的10X的解决方案的股票由提供者推荐的缓冲器中，并储存在-80℃下大多数ECM蛋白溶于去离子水，但可能需要进行调整，与滴乙酸的pH值。大多数生长因子，细胞因子，和用BSA的PBS中制备的重组受体的胞外结构域，但制造商的条件会有所不同。筛选的蛋白质通过0.45μm的4毫米尼龙针筒式过滤器（的Nalgene）之前存储的等分试样。 </li><li>设计一个主板根据所需蛋白的组合和稀释。通常依赖于贴壁细胞的至少一个兼容的ECM来介导细胞粘附的存在下，但也可以介导细胞表面抗原决定簇的抗体附着有时。这是一个好主意，添加FITC染料，或fluorphore结合蛋白的至少一个孔，以便阵列可以很容易地面向。 </li><li>通过稀释的蛋白质与印刷缓冲液的组合组成的100mM准备主板Tris-acetate/20％glycerol/0.05％，：TRITON-100X pH值5.2。通常情况下的384孔板的每个孔中包含不超过10微升。 </li><li>记录的内容以及每个制表符分隔的数据库文件中的每个主控板和各主控板提供了一个独特的识别号码。其次是设计师的缩写，一个六位数字的日期往往是的目的（MMDDYYinitial）。由于阱的体积是小的，蛋白质的等分试样可用于有效地生成大量复制板。建议主板被存储在-80℃和每个主板应接受不超过两个冻融循环。 </li></ol> 3。 MEArray印刷<ol><li> MEArrays可以打印最传统的芯片印刷的机器人。导盲犬小Q针打印机，可以使用硅或不锈钢针工作，但蛋白质的粘度可能会出现问题。毛细管打印机是理想的微阵列打印此应用程序的机器人，因为他们的工作以及含有粘性蛋白的解决方案。 </li><li>为了达到良好的统计力量在阵列中，10至12复制每个微点建议。这样的设计允许的活动在一个微相对于另一个在同一阵列中使用简单的T检验统计量的比较。 dunnette的测试可用于与其他微环境的控制环境中的活性进行比较。这样的设计最适合的功能型控制微环境已与之前执行MEArray实验。 </li><li>湿度应保持在50％左右。湿度控制是非常重要的，因为低湿度可以干的内销或主板造成效率低下的印刷基质沉积在井的解决方案。湿度可以有效地控制通过悬垂机器人与非多孔塑料布和同时使用设置，以保持50％的湿度的加湿器和加湿器。冷却的的印刷plattens可以是大家有用UL保留一些蛋白质，但一定要小心，以避免水汽凝结形成的幻灯片。 </li><li>打印每个数组应标有防冷冻幻灯片标签编码的序列号，由主控板的标识符，后面跟一个三位数（MMDDYYinitial-NNN）。由于每一个阵列使用或分发，其试验性治疗的细节，应保持在一个数据库中。跟踪的印刷日期和冻融循环主板将有助于确定最佳的条件下保持重现性。 </li><li>可以在-20℃储存在密封的滑动框印刷MEArrays应不超过1个月。重现性明显下降之后。 </li></ol> 4。上MEArrays培养哺乳动物细胞功能分析<ol><li>将文化室：细胞所需文化的MEArrays媒体和数字量的限制，塑料的腔为fitted环绕在印刷的阵列。对于许多阵列，从2室滑动（Nunc）中包含4.2厘米2的区域，可以使用一个单一的腔室。删除腔的制造室幻灯片，用刀片削减了一半的室。使用3毫升注射器涂上薄薄珠粒水族馆硅酮（DAP）的的一个MEArray表面上的一个腔室，然后按边缘。避免将水族馆硅腔阵列上的功能。 </li><li>阻塞和漂洗：MEArrays需要以及漂洗，以除去未反应的单体，它可以是对细胞有毒性。如果使用PDMS-包被的载玻片，然后在区域之间的印刷功能首先需要被阻止与非防污涂层，以防止细胞粘附;孵育中的阵列1％的Pluronic F108（BASF）的水或2％BSA的在真空下的15分钟的水。 PA凝胶幻灯片不需要阻塞步骤。在所有的情况下，冲洗细胞培养基的数组三次五分钟（媒体的选择取决于时所使用的细胞，但使用抗生素，建议无论媒体或细胞）。 PA凝胶需要额外的孵育30 min，在的媒体补充水分的凝胶。 </li><li>手机附件四，五阵列可以装进一个15厘米无菌培养皿中。盖上培养皿盖阵列保持无菌。加入一半的最终的介质卷的MEArray媒体为10,000〜1,000,000个细胞/ ml的最终浓度加入细胞。细胞将附加到印刷的功能以不同的速率取决于组合物，在印刷的微环境。请的均匀附着，通过查看阵列阶段通过倒置显微镜，在15至20分钟的间隔。通过轻轻摇动MEArrays来回，细胞附着在图案化的方式，可以区分从浮动的，未附着的细胞。 </li><li>除去未结合的细胞：在PA涂层MEArrays，未结合的细胞可以被吸入，并与适当的音量，可更换媒体。在PDMS-涂层MEArrays，媒体永远不能完全从井里取出，因为细胞几乎立刻就干死了。因此对PDMS-涂层MEArrays，未结合的细胞，必须除去的过程的连续介质的体积的一半交流，直到除去任何未结合的细胞，通过显微镜检查来确定。反润湿效果的PDMS是不太突出时定义的媒体相比，使用含血清的介质，并且，当使用BSA被阻止的未打印区域PLURONICS F108相比。 </li><li>放在15厘米的培养皿中的天数与正常媒体变化MEArrays细胞可以培养。媒体上PDMS幻灯片必须改变连续变化的媒体量的一半。 </li><li>普通的固定剂，如多聚甲醛和甲醇/丙酮，与MEArray系统兼容。上的PA，涂MEArrays的染色细胞时，固色剂可添加冲走，就像他们在常规染色过程。然而，当对PDMS涂覆MEArrays染色细胞，表面必须保持湿润，即使在固定。吸一半的媒体和更换用固色剂。重复几次，直到井的过程充满了大部分的固色剂。固定液固定后，正在逐渐取代用封闭缓冲液中相同的方式，是合适的下一个步骤，分析。 </li><li>常用的免疫组化染色来分析细胞的功能。染色例程将有所不同，但工作时，上的PDMS MEArrays，执行每一个需要洗涤和抽吸上述的步骤，逐渐改变的解决方案，绝不允许脱湿的表面。反润湿会导致染色的文物。 </li><li>的腔室，可以除去与援助剃刀刀片。盖玻片可以安装之上使用Fluoromount-G（Southern Biotech公司）染色MEArrays。可进行多色荧光微阵列扫描仪检测共焦显微镜电动平铺图像采集模式。 </li></ol> 5。代表性的成果图案化的蛋白沉积的一个例子，在一个印刷PDMS-优哉MEArray使用方嘴硅鹅毛笔销微阵列印刷机器人是在图2中所示的标签。印刷的各种蛋白质的沉积可以验证通过免疫荧光抗体（ 图2A）。在所述靠模样板的蛋白质溶液的稀释度的反射印刷基质的表面（ 图2B）上沉积的量（荧光强度）。细胞应附加到一个明显的图案化的方式（ 图2C）中的打印功能。 一个例子的一个MEArray实验示出，逆稀释两个微环境蛋白引起特定的角蛋白的表达谱中的蛋白质浓度依赖性的方式在一个人多能乳腺上皮progenitor细胞株（D920细胞），在图3中所示。气泡图是有用的，用于确定特定的表型是否强加于稀释系列的复制功能的细胞上。比如，如果一个特定的分子中的微环境，导致明显的表型，一旦指导成分已稀释够成的背景下进行的中性的ECM的表型应改变或消失。轴突4200A（Molecular Devices公司）微阵列扫描仪的角蛋白和角蛋白14的中间丝蛋白进行免疫荧光检测。十二复制稀释系列印在每个MEArray内，和角蛋白8比2的日志角蛋白14的平均荧光强度作为气泡图作图，得到真实的想法的信号的变化和重复性。示出的数据从一个MEArray细胞附着后，洗去未结合的细胞（ 图3A）是固定的，和24小时后的丘尔重（ 图3B）。这个相对较小的分析，使用了一个-way ANOVA分析，以确定在每个时间点的平均信号的方差，和分组的双尾T检验被用来确定是否不同稀释度的I型胶原蛋白和重组人P-钙粘蛋白引起的角蛋白表达的变化。有没有变化，但细胞的功能，只是附件中的平均24小时后接触​​到不同的微环境中的角蛋白表达细胞间有显着差异。 T-测试验证，I型胶原蛋白的浓度引起更高的角蛋白8的表达，而P-钙粘蛋白的浓度高引起了强烈的角蛋白14信号，24小时后。这样的结果是一致的，与以前的报告中，P-钙粘蛋白的微环境，将处以K14表达肌上皮细胞的表型双有力的乳腺祖细胞4。 一个例子的整个scanne“ðMEArray印刷上的40000帕PA凝胶是在图4中所示。 <img alt="图1" src="/files/ftp_upload/4152/4152fig1.jpg" /> 图1。甲的流程图的MEArray程序。首先，印刷基质制备与PDMS或PA。二，主板准备和在数据库中注明。第三，MEArrays印刷和编码的序列号。第四，培养室连接，表面是块和/或冲洗，然后细胞允许附加和未结合的细胞被冲走。第五，细胞可以用染色或生物测定培养一段时间后，根据实验设计。最后，画像的MEArray可以以下方式获得，并与合适的扫描器和软件分析。 <img alt="图2" src="/files/ftp_upload/4152/4152fig2.jpg" /> 图2。沉积，银行足球比赛和相对丰富的印刷蛋白质可以验证之前与免疫组化的细胞附着。 A）抗体确认Ⅳ型胶原蛋白和层粘连蛋白-111被用来验证他们的印刷的MEArray功能的存在。 B）使用在NIH ImageJ软件，整个一系列的稀释，开始从200微克/毫升的蛋白质溶液的两种蛋白质的相对丰度的平均像素强度的分析功能，可以定性评估。 C）第二阶段D920细胞的显微照片PDMS涂层的印刷MEArray方形特征。 <img alt="图3" src="/files/ftp_upload/4152/4152fig3.jpg" /> 图3，使用角蛋白表达的变化作为一个功能的时间和微环境中的多能祖细胞系的一个MEArray分析的一个例子。每个气泡代表比例的角蛋白和角蛋白14蛋白水平从10-15细胞附着的有限元分析TURE在MEArray。表达用免疫荧光探针。 A）显示细胞角蛋白的比率只是安装后，和B）示出在24小时后的阵列上，镀平行的角蛋白比率。两种蛋白质的最大浓度为200μg/ ml和稀释2倍。气泡的直径表示日志2比角蛋白8和角蛋白14的平均强度的幅度，并且橙色和白色的颜色编码指示值&gt; 0和&lt;0，分别。 F值单向ANOVA和P值从T-测试，和括号内的带箭头的确定种群相比，被示出。 <img alt="图4" src="/files/ftp_upload/4152/4152fig4.jpg" /> 图4。一个例子收购的MEArray扫描，在激光扫描共聚焦显微镜使用瓷砖收购的模式。 HCC1569细胞，我们可以将DNA模拟的知识，固定前4小时。DAPI（蓝色）和教育（红色）。

<ol>
	<li>Bissell, M. J., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape,+cytoskeletal+tension,+and+RhoA+regulate+stem+cell+lineage+commitment.">Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.</a> Dev. Cell. 6, 483-495 (2004).</li><li>LaBarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+progenitor+cell+fate+decsions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decsions are products of interactions with combinatorial microenvironments.</a> Integrative Biology. 1, 70-79 (2009).</li><li>Kim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Methods+for+Polymers+in+Cell+and+Tissue+Engineering.">Patterning Methods for Polymers in Cell and Tissue Engineering.</a> Annals of biomedical engineering. , (2012).</li><li>Boudou, T., Ohayon, J., Picart, C., Pettigrew, R. I., Tracqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+elastic+properties+of+polyacrylamide+gels:+implications+for+quantification+of+cellular+forces.">Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces.</a> Biorheology. 46, 191-205 (2009).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current protocols in cell biology. Chapter 10, Unit 10 (2010).</li><li>Flaim, C. J., Chien, S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+matrix+microarray+for+probing+cellular+differentiation.">An extracellular matrix microarray for probing cellular differentiation.</a> Nat. Methods. 2, 119-125 (2005).</li><li>Soen, Y., Mori, A., Palmer, T. D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+regulation+of+human+neural+precursor+cell+differentiation+using+arrays+of+signaling+microenvironments.">Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.</a> Mol. Syst. Biol. 2, 37 (2006).</li></ol>

没有利益冲突的声明。

MEArray这里介绍的方法使细胞的组合微环境的相互作用4的功能分析。 MEArray分析相结合的基本缩微技术，细胞生物学，基因芯片印刷的机器人和分析设备，可在许多多用户设施的使用。 MEArray屏幕是与最贴壁细胞类型兼容，虽然在某些情况下，可能需要进行调整，无血清培养基配方包括BSA或&lt;1％血清，从而可以提高附着。该方法只限于由试剂用于分析一个给定的细胞功能的可用性，基于?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录号 </td><td> 评论（可选） </td></tr><tr><td>载玻片25毫米×75毫米</td><td> VWR </td><td> 48311-600 </td><td></td></tr><tr><td> 24毫米×50毫米盖玻片（第1号） </td><td> VWR </td><td> 48393-241 </td><td></td></tr><tr><td>染色盘（或Coplan JAR） </td><td> VWR </td><td> 25461-003 </td><td></td></tr><tr><td>皮氏培养皿（15厘米） </td><td> BD猎鹰</td><td> 351058 </td><td></td></tr><tr><td> NaOH溶液（1.0N） </td><td> Sigma-Aldrich公司</td><td> S2567 </td><td></td></tr><tr><td> APES的（&gt; 98％（3 - 氨基丙基）三乙氧基硅烷） </td><td> Sigma-Aldrich公司</td><td> A3648 </td><td></td></tr><tr><td>戊二醛</td><td> Sigma-Aldrich公司</td><td> G7651 </td><td> 50％水</td></tr><tr><td> APS（&gt; 98％过硫酸铵） </td><td>小号IGMA公司</td><td> A3678 </td><td>准备10％的工作DDH 2 O </td></tr><tr><td> TEMED（N，N，N&#39;，N&#39;-四甲基乙二胺） </td><td> Sigma-Aldrich公司</td><td> T9281 </td><td></td></tr><tr><td>丙烯酰胺（40％） </td><td> Sigma-Aldrich公司</td><td> A4058 </td><td></td></tr><tr><td>双 - 丙烯酰胺（2％w / v的） </td><td>费舍尔生物</td><td> BP1404-250 </td><td></td></tr><tr><td> 0.45微米针头式过滤器4毫米的尼龙</td><td>的Nalgene </td><td> 176-0045 </td><td></td></tr><tr><td> FITC </td><td> Sigma-Aldrich公司</td><td> F4274 </td><td></td></tr><tr><td> PDMS（聚二甲基硅氧烷） </td><td>道康宁</td><td> 3097358-1004 </td><td> Sylgard的184弹性体套件，通过安士澳粘合剂</td></tr><tr><td> 2 - 室玻片</td><td> NUNC </td><td> 177380 </td><td></td></tr><tr><td>普朗尼克F108 </td><td>巴斯夫</td><td> 30089186 </td><td></td></tr><tr><td> AQUarium密封胶</td><td>道康宁</td><td> DAP 00688 </td><td></td></tr><tr><td> Fluormount-G </td><td>南方生物技术</td><td> 0100-01 </td><td></td></tr><tr><td>一次性塑料杯</td><td></td><td></td><td></td></tr><tr><td>压舌板</td><td></td><td></td><td></td></tr><tr><td>丁腈手套</td><td></td><td></td><td></td></tr><tr><td>塑料显微镜载物片盒</td><td></td><td></td><td></td></tr><tr><td>旋转涂布机</td><td> WS-400B-6NPP/LITE </td><td>劳雷尔技术公司</td><td></td></tr><tr><td>烤箱</td><td></td><td></td><td></td></tr><tr><td>数码烤盘</td><td></td><td></td><td></td></tr><tr><td> 384 - 孔板</td><td></td><td></td><td>一个品牌的合适的芯片机器人</td></tr><tr><td>微阵列印刷机械</td><td></td><td></td><td></td></tr><tr><td>倒相和荧光显微镜</td><td></td><td></td><td></td></tr><tr><td>轴突微阵列扫描仪</td><td> Molecular Devices公司</td><td></td><td>存在多种配置</td></tr></tbody></table>

fabrication and use of microenvironment microarrays (mearrays)

细胞和其周围的微环境之间的相互作用，有功能的细胞行为的后果。在单细胞水平上，不同的微环境，可以并处分化，迁移和增殖表型，并在组织水平上的微处理复杂的形态发生和肿瘤发生1。不仅做的细胞和分子影响细胞的微环境内，但这样做的组织的弹性和几何。细胞 - 细胞 - 细胞外基质，和可溶性因子交互作用的总和定义为，在除了物理特性，在微环境是复杂的。内的组织的细胞的表型是部分由于它们的基因组的内容和部分由于与微环境的组合的相互作用。一个主要挑战是连接特定组合的微环境的组成部分，具有鲜明的行为。 ENT“&gt;在这里，我们提出基于细胞的功能筛选的组合微环境的相互作用与4的微芯片（MEArray”）平台。该方法可同时控制的分子组成和弹性模量，并结合广泛使用的芯片的使用和缩微技术。MEArray屏幕需要最少为10,000细胞，每个阵列，这有利于功能研究罕见的细胞类型，如成年祖细胞。该技术的一个限制是，整个组织的微环境可以不被完全扼要重述上MEArrays。然而，比较反应在相同的细胞类型中的大量相关的微环境，例如成对表征一个给定的组织的细胞外基质蛋白的组合，将提供如何微环境的组件引起组织特异性的功能表型的洞察。 可以使用各种不同的重组组打印MEArrays一族因子，细胞因子，和纯化的细胞外基质蛋白，以及它们的组合。该平台被限制仅由特定试剂的可用性。 MEArrays经得起时间推移分析，但最经常使用的细胞功能，可测量与荧光探针的终点分析。例如，常用的测量DNA的合成，细胞凋亡，收购的分化状态，或特定基因产物的生产。简单地说，这个基本流程的MEArray实验是准备与印刷基质涂覆的滑动，并准备要打印的蛋白质，是主板。然后阵列都印有微阵列机器人，允许细胞附着，生长在培养，然后进行化学固定在到达实验终点。用于成像的荧光或比色测 ​​定法，与传统的显微镜或微阵列扫描仪，以显示相关的分子和细胞的表型（ 图1）。

Watch this Scientific Journal Video about Fabrication and Use of MicroEnvironment microArrays MEArrays at JoVE.com

Fabrication and Use of MicroEnvironment microArrays MEArrays

一个组合功能性筛选方法获得洞察的分子组成的微环境对细胞功能的影响。该方法充分利用了现有的基于微阵列技术来生成数组的定义组合的微环境，支持细胞的粘附和功能分析。

制造和使用微环境芯片（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

fabrication-and-use-of-microenvironment-microarrays-mearrays

Research

JoVE Journal

Biology

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

9.9K 次观看.  Lawrence Berkeley National Laboratory. 一个组合功能性筛选方法获得洞察的分子组成的微环境对细胞功能的影响。该方法充分利用了现有的基于微阵列技术来生成数组的定义组合的微环境，支持细胞的粘附和功能分析。

视频: Fabrication and Use of MicroEnvironment microArrays MEArrays

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

PDMS Device Fabrication and Surface Modification

PDMS的设备制造和表面改性

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

安培电极的制备

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

制作生肌工程的组织结构

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

（充电）酰化tRNA的使用微阵列技术水平的全基因组分析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA微阵列：样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

组织芯片，​​免疫组织化学染色和数字化生产在人类蛋白质图集

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

糖基谱研究植物细胞壁聚合物，用微阵列

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

复用的恒化器阵列的设计与应用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...