We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.

1.清洁玻璃表面和试剂的准备<ol><li>放置一个玻璃表面在氧等离子体机，在50％的功率进行清洁5分钟。 </li><li>制作2.5毫升2-％重构（3-氨基丙基）三乙氧基硅烷（APTES）溶液中，加入APTES 50微升并在锥形管2.45毫升乙醇中。 </li></ol> 2. APTES和DSB功能化<ol><li>添加APTES溶液到表面。每孔吸取150微升8孔板。每孔移液器将100μl24孔板中。吸取1.1毫升60×15毫米的玻璃菜肴。覆盖表面，防止蒸发和APTES解决分配不均。放置在表面上的平台摇动器在室温下50分钟，它创建一个连APTES层的分布。 注:APTES是形成表面的第一层的氨基硅烷。如果使用不同的表面，启发式决定的，以覆盖表面所需溶液的体积。 </li><li>选择用于烤箱的温度，而表面上的摇床:热至55℃2小时的8孔平板和24孔平板中。热玻璃只菜至90℃1小时。 </li><li>冲洗用乙醇的表面上。 <ol><li>辖的乙醇通过参考根据所用的表面上所需的量。添加150微升乙醇至每个孔8孔板中。添加125微升乙醇至每个孔24孔板中。加入1.1毫升乙醇玻璃菜肴。 </li><li>通过排放和绘图从固定点的液体，如井的拐角冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。冲洗用乙醇两次。 注意:如果任何玻璃底孔板的在其中有任何塑料，不加热它们高于65℃时，由于塑料会开始熔化和变形。 </li></ol></li><li>干与来自罐分配100％的氮气。放置在表面的OVEN。 </li><li>通过在37.5微升二甲基亚砜（DMSO）的1.5毫克/毫升的DSB组合在2462.5制备2.5的d脱硫生物素（DSB）溶液中，和5毫克/毫升的1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）的0.1M的4-吗啉乙磺酸水合物（MES）（pH值为6）缓冲液的微升。然后结合这两种解决方案。 </li><li>加入1μl2-β巯基乙醇，15分钟后，在溶液中以骤冷DSB和EDC之间的反应。删除从烤箱功能化玻璃表面热APTES。等待5-10分钟的玻璃表面冷却。 </li><li>添加MES缓冲表面冲洗，使用基于表面上的金额。添加150微升的MES缓冲液，以每孔8孔板中。添加125微升的MES缓冲液，以每孔24孔板中。加入1.1毫升MES缓冲玻璃菜肴。 </li><li>通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向入面。冲洗更多的MES缓冲两次。 </li><li>申请所述DSB溶液到表面，以允许他们孵育。加入每孔150微升DSB解决方案8孔板。添加每孔100μl的DSB溶液中24孔板中。添加1.1毫升玻璃菜DSB解决方案。 </li><li>放置DSB覆盖玻璃表面上的培养皿内的湿纸巾。盖上盖子，在4℃冰箱孵育18-24小时。 </li></ol> 3.链霉亲和功能化<ol><li>冲洗每个玻璃表面三次用1ml 1.0倍磷酸盐缓冲盐水（PBS）中的。通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。稀释链亲和素（SAV）原液0.4毫克/毫升（推荐）。 注:PBS等渗因此它可以用作用于稀释和冲洗的介质。 PBS被用作为SA的溶剂v和因此，不会影响到SAV的结合到表面的能力。 </li><li>均匀地涂0.4毫克/毫升的SAV溶液到表面，使得在玻璃的底部的形式的薄层，选择的溶液基于表面的量。对于8孔板，每孔加入150微升0.4毫克/毫升SAV解决方案。对于24孔板，每孔加入100微升0.4毫克/毫升SAV解决方案。对于玻璃菜，加1.1毫升0.4毫克/毫升SAV解决方案。 </li><li>覆盖和移动板到14厘米培养皿中保持水分。孵育在冰箱培养皿18-24小时。 注:重要的是要利用每一个表面上的APTES，DSB和SAV一致卷。 </li><li>冲洗每个玻璃表面用150微升的PBS三次以除去SAV。通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。 </li><li>湿纸巾W¯¯第i个去离子水，并放置在纸巾平在围绕板中的孔中，以保持水分14厘米培养皿。覆盖包含井培养皿。直到需要孵化培养皿在4℃生物安全水平1（BSL-1）的冰箱。 </li></ol> 4.细胞捕获和释放<ol><li>开始与旨在用于实验的细胞T175烧瓶（多个）。从烧瓶（多个）吸媒体。洗出剩余介质用5ml室温PBS中。抽吸PBS从烧瓶（多个）。 </li><li>加入10 mL的非酶起重剂，如细胞解离溶液，对细胞的T-175烧瓶中。把该烧瓶在孵化6分钟，以允许从烧瓶的细胞的升降。 </li><li> 6分钟后，加入10毫升冷汉克的平衡盐溶液（HBSS;见材料清单）灭活解除代理。取出20微升细胞计数使用血细胞计数器细胞在溶液中的数量。 注:根据FUNCtionalized在捕获实验中所用的表面，所需的细胞数量不同而不同。对于官能8孔板，使用每孔30万细胞。对于功能化玻璃餐具，使用110万细胞。对于官能化24孔板，推荐125000细胞。对细胞计数的更多信息补充协议中找到。 </li><li>重复步骤4.1- 4.3为细胞另一个烧瓶中，如果较少的细胞存在比需要的实验。结合细胞悬浮液和验票细胞获得在溶液中的细胞总数。 </li><li>降速细胞悬浮液在离心机中，在500×g离心5分钟，在4℃以获得浓缩的细胞的沉淀。找到的HBSS适量添加到细胞悬浮液以获得每毫升1×10 6个细胞的浓度，然后从纺丝向下细胞吸出上清液，并添加计算量。这个体积可使用在补充的公式计算。 </li><li>移液器上下（磨碎）重悬日E细胞在溶液中并且降低的解决方案，可以减少抗体结合的细胞聚集。拆分移动解决方案到独立的控制和实验方案。查看组件和计算补充文件。 </li><li>添加生物素化的抗体在补充文件描述的各个小区的解决方案。在4℃孵育30分钟上的端过端混频器。 注:对于MCF7GFP细胞，0.5毫克/毫升hIgG或0.5毫克/毫升HLA-ABC抗体建议。对于RAW巨噬细胞，1毫克/毫升mCD11b以一半体积推荐占稀释差异。对于人脐静脉内皮细胞（HUVEC），0.5毫克/推荐毫升hCD31。 </li><li>用HBSS洗官能玻璃表面。对于这个实验中，一个8孔板被劝告。 <ol><li>添加150微升HBSS至每个孔8孔板中。用HBSS清洗两次。通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。冷保存在4℃。 </li></ol></li><li>添加细胞溶液至孔，等待45分钟的细胞上的振动筛冰上孵育。通过使用如在补充所述计算的体积使得在无菌HBSS生物素的溶液中。 </li><li>通过使用HBSS除去从玻璃井细胞溶液。 <ol><li>轻轻吸取150微升HBSS到每个孔中。然后吸取了HBSS。通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。 </li><li>重复两次以上。清洗后，加入HBSS到孔，以保持湿润的细胞。 </li></ol></li><li>加入150微升20毫米生物素溶液各有关释放好，然后等待20分钟，以便反应。 </li><li>回忆非特异性结合的细胞如上所述S按用HBSS洗涤，然后如果使用荧光标记的细胞，进行荧光图像的细胞。此外，在一烧瓶图像活细胞（作为对照，以与孔中的细胞比较）。如果使用绿色荧光蛋白（GFP）转染的细胞中，激励将470纳米，和发射将515纳米。 </li></ol> 5.抗体优化:抗体滴定<ol><li>通过解除从T75或T175烧瓶中的细胞作为在步骤4.1- 4.3解释开始。 注意:这些卷校准24孔板，但可以改变，以适应通过启发式检测任何玻璃官能表面。代表性抗体滴定如图1所示。 </li><li>算使用血细胞计数器细胞，然后在500 xg离心在4℃下降速细胞溶液中的圆锥形管5分钟。重组细胞每毫升1-百万细胞，使用在补充所述的计算。 </li><li>拆分1米100万年细胞每毫升溶液注入500微升在不同离心管的抗体（抗体）溶液六个不同的解决方案。 <ol><li>稀释抗体原液至10微克/毫升，1微克/毫升，100纳克/毫升，10纳克/毫升，1毫微克/毫升。通过使用100微升染色缓冲液（PBS + 1％叠氮化钠+ 1％BSA）中，并加入500μl的细胞与没有抗体在它的控制创建控件。对于空白对照（无Ab和无细胞），使用300微升的PBS中的溶液。 注意:注意:叠氮化钠是剧毒，易爆，并格外小心，必须使用时服用。请参考材料安全数据表（MSDS），并使用适当的安全设备。 </li></ol></li><li>结合，并在4℃下孵育，在最终过端混合器的电池溶液的抗体溶液30分钟。冲洗用HBSS三次官能24孔板中。通过排放和绘图从固定点的液体，如井的拐角冲洗表面。按住PI佩特以大约70°角，以便尖端不直接指向到表面。 </li><li>分装125微升每种样品溶液到相应的APTES，DSB和SAV功能化的很好。孵育在4℃下或在冰上在玻璃表面上45分钟。用HBSS冲洗表面三次以除去非特异性附着的细胞。通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。不要冲洗底部的行，因为这些是对照。 </li><li>添加150微升的HBSS每个充分洗涤后，把24孔平板在冰上，然后用读板器测量GFP细胞（励磁485纳米/发射528纳米）的荧光。 </li></ol> 6.单元优化:细胞滴定<ol><li>提起细胞步骤4.1 -4.3提及。 </li><li>算使用血细胞计数器细胞。降速细胞等等lution在500×g离心5分钟，锥形管在4℃下。重组细胞每毫升1-百万个细胞。 注:更多关于使用血细胞计数器的信息可以在补充协议中找到。 </li><li>吸取从锥形管的160万股票的细胞和地点细胞溶液1.6毫升。从锥形管800,000细胞股票和地点细胞溶液吸取800微升细胞。吸管从细胞股票80微升了在锥形管80,000个细胞股票和地点。移液器从8000细胞股票和地点细胞股票8微升的锥形管。 注:浓度是每8孔板测量给出，根据需要进行复制。板输出的一例示于图2。 </li><li>采取四种储备液，并在4℃下以500 xg离心在离心机中旋转5分钟。重新暂停所有股票的解决方案，400微升HBSS的。 </li><li>加入1.6微升100毫微克/毫升抗体，以1.6亿的股票，0.8μl至80万细胞原液，0.5微升到所有其他股票。温育30分钟，抗体在最终过端混合器在4℃，30分钟。洗用HBSS三倍官能玻璃表面。 注:建议显微镜定量的官能8孔板，同时建议酶标仪定量功能化的24孔板。对于8000细胞库存抗体浓度没有降低，以允许捕获表面的限制因素，以不缺乏溶液抗体，而是表面的捕获性能。 </li><li>应用150微升样品溶液的各孔中。应用160万细胞库存到孔在左边的第一列。申请800,000细胞库存从左侧的第二列。申请80,000细胞库存从左侧第三列。最后套用8000细胞股票的最后一列。在4℃下孵育45分钟的摇动。 注:160万细胞库存作为待测试的最高浓度，并且是通常用于细胞分离的实验（每孔600,000个单元格）800,000细胞库存用作实验中的控制，因为它是在典型的蜂窝浓度细胞的双重量即在细胞分离实验中使用的（每孔3000000细胞）。 80000细胞库存用作稀释10倍，以测试较低浓度为蜂窝捕获（每孔30,000个细胞）。 8000细胞库存用作百倍稀释到作为测试来检查细胞的分离的下限（每孔3000个细胞）。 </li><li>用HBSS三次冲洗。通过排放和绘图从固定点的液体，如井的角落冲洗表面。握住吸管以大约70°角，以便尖端不直接指向到表面。收集所有的洗涤。 </li><li>图象上荧光显微镜的细胞，如果使用8孔板，取各拍照河畔面，并在4℃下在摇动器应用150微升生物素以每面为一小时。一小时后，冲洗生物素释放孔用HBSS 3倍之前。收集井全部用于洗涤与血球和图像释放井计数。 </li><li>应用150微升的20mM过量的生物素的溶液，以适当的生物素释放孔1小时，如果使用24孔板，然后用HBSS冲洗这些油井的3倍。定量用酶标仪24孔板中。使用血球到所有收集以及洗涤细胞计数。 </li></ol> 7.图像分析注意:斐济软件包（http://fiji.sc/Fiji）被推荐用于图像分析。最初，图像转换成灰度图像，然后将该亮度/对比度改变为带出的细胞。 <ol><li>加载图像，并转换通过单击图像选项卡，然后向下滚动到"类型"，然后单击灰度4，8位"。 </li><li>通过单击图像选项卡，然后滚动到"调整"增加图像的对比度。点击"亮度和对比度"，然后使用滚动条，使细胞中脱颖而出。如果使用荧光图像，反转图像，使更多的定义的细胞。 </li><li>加载插件ImageJ的上称为ITCN（http://rsb.info.nih.gov/ij/plugins/itcn.html）来分析图像。 </li><li>打开ITCN。当出现一个对话框，提示与多个参数的用户估计单元的尺寸，第一组感兴趣的细胞的最小宽度。如果图像是荧光灯和将细胞变暗点击"检测暗峰"选项。 </li><li>初始设定为2的阈值，然后打"计数"按钮。画面的一个新版本的计会出现红点划定在哪里细胞已被计算在内。 </li><li>调整阈值和宽度参数度日defini更准确的蜂窝数据纳克"小区"的大小。注意:计算将在右边的对话框细胞的数目。更改参数将给予不同数量的计数的细胞。 </li><li>继续通过阈值和宽度参数，直至达到对细胞数目的良好估计迭代。 </li></ol>

<ol>
	<li>Erdbruegger, U., Haubitz, M., Woywodt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+endothelial+cells:+a+novel+marker+of+endothelial+damage.">Circulating endothelial cells: a novel marker of endothelial damage.</a> Clin. Chim. Acta. 373 (1-2), 17-26 (2006).</li><li>De Spiegelaere, W., Cornillie, P., Van Poucke, M., Peelman, L., Burvenich, C., Van den Broeck, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mRNA+expression+analysis+in+kidney+glomeruli+using+microdissection+techniques.">Quantitative mRNA expression analysis in kidney glomeruli using microdissection techniques.</a> Histol. Histopathol. 26 (2), 267-275 (2011).</li><li>Chen, S., Guo, X., Imarenezor, O., Imoukhuede, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+VEGFRs,+NRP1,+and+PDGFRs+on+Endothelial+Cells+and+Fibroblasts+Reveals+Serum,+Intra-Family+Ligand,+and+Cross-Family+Ligand+Regulation.">Quantification of VEGFRs, NRP1, and PDGFRs on Endothelial Cells and Fibroblasts Reveals Serum, Intra-Family Ligand, and Cross-Family Ligand Regulation.</a> Cell. Mol. Bioeng. 8 (3), 383-403 (2015).</li><li>Cheung, L. S. L., 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=Detachment+of+captured+cancer+cells+under+flow+acceleration+in+a+bio-functionalized+microchannel.">Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel.</a> Lab Chip. 9 (12), 1721-1731 (2009).</li><li>Privorotskaya, 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=Rapid+thermal+lysis+of+cells+using+silicon-diamond+microcantilever+heaters.">Rapid thermal lysis of cells using silicon-diamond microcantilever heaters.</a> Lab Chip. 10 (9), 1135-1141 (2010).</li><li>Park, K., Akin, D., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+capture+and+lysis+of+vaccinia+virus+particles+using+silicon+nano-scale+probe+array.">Electrical capture and lysis of vaccinia virus particles using silicon nano-scale probe array.</a> Biomed. Microdevices. 9 (6), 877-883 (2007).</li><li>Galletti, G., Sung, M., Vahdat, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+breast+cancer+and+gastric+cancer+circulating+tumor+cells+by+use+of+an+anti+HER2-based+microfluidic+device.">Isolation of breast cancer and gastric cancer circulating tumor cells by use of an anti HER2-based microfluidic device.</a> Lab Chip. 14 (1), 147-156 (2014).</li><li>Schudel, B. R., Choi, C. J., Cunningham, B. T., Kenis, P. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chip+for+combinatorial+mixing+and+screening+of+assays.">Microfluidic chip for combinatorial mixing and screening of assays.</a> Lab Chip. 9 (12), 1676-1680 (2009).</li><li>Lien, K. Y., Chuang, Y. H., 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=Rapid+isolation+and+detection+of+cancer+cells+by+utilizing+integrated+microfluidic+systems.">Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems.</a> Lab Chip. 10 (21), 2875-2886 (2010).</li><li>Stott, S. L., 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=Isolation+of+circulating+tumor+cells+using+a.">Isolation of circulating tumor cells using a.</a> PNAS. 107 (35), 18392-18397 (2010).</li><li>Yu, M., Ting, D., Stott, S., Wittner, B., Ozsolak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+of+pancreatic+circulating+tumour+cells+implicates+WNT+signalling+in+metastasis.">RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis.</a> Nature. 487 (7408), 510-513 (2012).</li><li>Sheng, W., Ogunwobi, O., Chen, T., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=>Capture,+release+and+culture+of+circulating+tumor+cells+from+pancreatic+cancer+patients+using+an+enhanced+mixing+chip.">>Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.</a> Lab Chip. 14 (1), 89-98 (2014).</li><li>Zheng, X., Cheung, L. S. L., Schroeder, J. A., Jiang, L., Zohar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-performance+microsystem+for+isolating+circulating+tumor+cells.">A high-performance microsystem for isolating circulating tumor cells.</a> Lab Chip. 11 (19), 3269-3276 (2011).</li><li>Imoukhuede, P. I., Popel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+cell-to-cell+variation+of+vascular+endothelial+growth+factor+receptors.">Quantification and cell-to-cell variation of vascular endothelial growth factor receptors.</a> Exp. Cell Res. 317 (7), 955-965 (2011).</li><li>Imoukhuede, P. I., Popel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+VEGF+receptors+on+endothelial+cells+in+mouse+skeletal+muscle.">Expression of VEGF receptors on endothelial cells in mouse skeletal muscle.</a> PLoS One. 7 (9), e44791 (2012).</li><li>Ludwig, A., Kretzmer, G., Sch&#252;gerl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+a+&amp;#34;critical+shear+stress+level&amp;#34;+applied+to+adherent+mammalian+cells.">Determination of a &amp;#34;critical shear stress level&amp;#34; applied to adherent mammalian cells.</a> Enzyme Microb. Technol. 14 (3), 209-213 (1992).</li><li>Ansari, A., Lee-Montiel, F. T., Amos, J., Imoukhuede, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+anchor+targeted+cell+release.">Secondary anchor targeted cell release.</a> Biotechnol. Bioeng. 112 (11), 2214-2227 (2015).</li><li>Drenan, R. M., Nashmi, R., Imoukhuede, P., Just, H., McKinney, S., Lester, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+trafficking,+pentameric+assembly,+and+subunit+stoichiometry+of+neuronal+nicotinic+acetylcholine+receptors+containing+fluorescently+labeled+alpha6+and+beta3+subunits.">Subcellular trafficking, pentameric assembly, and subunit stoichiometry of neuronal nicotinic acetylcholine receptors containing fluorescently labeled alpha6 and beta3 subunits.</a> Mol. Pharmacol. 73 (1), 27-41 (2008).</li><li>Imoukhuede, P. I., Dokun, A. O., Annex, B. H., Popel, A. 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-by-cell+profiling+reveals+temporal+dynamics+of+VEGFR1+and+VEGFR2+membrane-localization+following+murine+hindlimb+ischemia.">Endothelial cell-by-cell profiling reveals temporal dynamics of VEGFR1 and VEGFR2 membrane-localization following murine hindlimb ischemia.</a> Am J Physiol Hear. Circ Physiol. 4 (8), H1085-H1093 (2013).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., van der Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3 (6), 1085-1091 (2008).</li><li>Imoukhuede, P. I., Popel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescent+profiling+of+VEGFRs+reveals+tumor+cell+and+endothelial+cell+heterogeneity+in+breast+cancer+xenografts.">Quantitative fluorescent profiling of VEGFRs reveals tumor cell and endothelial cell heterogeneity in breast cancer xenografts.</a> Cancer Med. 3 (2), 225-244 (2014).</li><li>BD Biosciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CD Marker Handbook: Human and Mouse. , (2010).</li><li>Tanzeglock, T., Soos, M., Stephanopoulos, G., Morbidelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammalian+cell+death+by+simple+shear+and+extensional+flows.">Induction of mammalian cell death by simple shear and extensional flows.</a> Biotechnol. Bioeng. 104 (2), 360-370 (2009).</li><li>Perritt, D., Wong, P., Macpherson, J. L., Henrichsen, K., Symonds, G., Pond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Processing Blood. , (2014).</li><li>Fukuda, S., 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=Centrifugation+attenuates+the+fluid+shear+response+of+circulating+leukocytes.">Centrifugation attenuates the fluid shear response of circulating leukocytes.</a> J. Leukoc. Biol. 72 (July), 133-139 (2002).</li><li>dela Paz, N. G., Walshe, T. E., Leach, L. L., Saint-Geniez, M., D'Amore, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+shear-stress-induced+VEGF+expression+in+endothelial+cell+survival.">Role of shear-stress-induced VEGF expression in endothelial cell survival.</a> J. Cell Sci. 125 (Pt 4), 831-843 (2012).</li><li>Allard, W. J., 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=Tumor+Cells+Circulate+in+the+Peripheral+Blood+of+All+Major+Carcinomas+but+not+in+Healthy+Subjects+or+Patients+With+Nonmalignant+Diseases+Tumor+Cells+Circulate+in+the+Peripheral+Blood+of+All+Major+Carcinomas+but+not+in+Healthy+Subjects+or+Patients+With+Nonmalignant+diseases.&amp;#34.">Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant diseases.&amp;#34.</a> Clinical Cancer Research. 10, 6897-6904 (2005).</li><li>Nagrath, S., 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=Isolation+of+rare+circulating+tumour+cells+in+cancer+patients+by+microchip+technology.">Isolation of rare circulating tumour cells in cancer patients by microchip technology.</a> Nature. 450 (7173), 1235-1239 (2007).</li><li>Chen, S., Weddel, J., Gupta, P., Conard, G., Parkin, J., Imoukhuede, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QFlow+Cytometer-Based+Receptoromic+Screening:+A+High-throughput+Quantification+Approach+Informing+Biomarker+Selection+and+Nanosensor.">QFlow Cytometer-Based Receptoromic Screening: A High-throughput Quantification Approach Informing Biomarker Selection and Nanosensor.</a> Submiss. , (2016).</li><li>Vasa, 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=Number+and+migratory+activity+of+circulating+endothelial+progenitor+cells+inversely+correlate+with+risk+factors+for+coronary+artery+disease.">Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease.</a> Circ. Res. 89 (1), E1-E7 (2001).</li><li>Hirsch, J. D., Eslamizar, L., 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=Easily+reversible+desthiobiotin+binding+to+streptavidin,+avidin,+and+other+biotin-binding+proteins:+uses+for+protein+labeling,+detection,+and+isolation.">Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation.</a> Anal. Biochem. 308 (2), 343-357 (2002).</li><li>Wilchek, M., Bayer, E. 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+Avidin-Biotin+Technology:+Literature+Survey.">Applications of Avidin-Biotin Technology: Literature Survey.</a> Methods Enzymol. 152 (1), 183-189 (1987).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescent+labeling+of+cancer+marker+Her2+and+other+cellular+targets+with+semiconductor+quantum+dots.">Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.</a> Nat. Biotechnol. 21 (1), 41-46 (2002).</li><li>Lee-Montiel, F. T., Imoukhuede, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+quantum+dot+calibration+standards+for+quantitative+fluorescent+profiling.">Engineering quantum dot calibration standards for quantitative fluorescent profiling.</a> J. Mater. Chem. B. 1, 6434 (2013).</li><li>Hornes, E., Korsnes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oligonucleotide-linked magnetic particles and uses thereof. , (1996).</li><li>Naranbhai, V., 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=Impact+of+blood+processing+variations+on+natural+killer+cell+frequency,+activation,+chemokine+receptor+expression+and+function.">Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function.</a> J. Immunol. Methods. 366 (1-2), 28-35 (2011).</li><li>Yadav, A. R., Sriram, R., Carter, J. A., Miller, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+solution-phase+and+vapor-phase+deposition+of+aminosilanes+on+silicon+dioxide+surfaces.">Comparative study of solution-phase and vapor-phase deposition of aminosilanes on silicon dioxide surfaces.</a> Mater. Sci. Eng. C. 35 (1), 283-290 (2014).</li></ol>

The authors have nothing to disclose.

在细胞分离技术的进步，进一步加强结构与功能的关系科学的研究在神经科学18，遏制再生生物学血管生物学19单元编程和血管生成的信号。事实上，原代细胞培养20（ 例如 ，内皮细胞）在血管生物学主要通过使用细胞分离技术进行。细胞分离最近还被用于细胞膜受体3,14,15,19,21定量流量（qFlow）仪分析。但是，现有的细胞分离方法，影响细胞表面受体的水平...

当前台式细胞分离方法（ 如荧光活化细胞分选1，激光捕获显微解剖2，免疫磁珠分离1）可能需要几个小时的准备和排序的。这些大的时间尺度可影响生理反应和表达水平，导致在不能代表生理反应3的分析。系统需要能够迅速和有效地分离出特定的细胞类型，而为了提高生物医学应用细胞分离和富集破坏细胞表面受体的水平。因此，我们的做法的理由是开发细胞分离温和的做法。 该"芯片实验室"的理念，提供更快的幅度（小时到分钟）细胞分离的订单的承诺，最经常涉及捕捉到细胞表面和释放细胞或细胞内的小故事通过物理4,5 NTS或化学方法6。虽然这些方法提供了一些优点，如鉴定蛋白质7,8-表达，识别RNA表达9-11，或甚至提供用于在体外培养12,13细胞，许多这些技术不能被转换为诊断如细胞受体分析因到它们的非生理环境。酶促起重剂如胶原酶也可影响这些受体数量14,15，这意味着使用这些起重剂不会产生准确的生理数据细胞受体定 ​​量技术。细胞裂解防止分化的原始表面受体之间，以及那些以前内在16。本协议描述了细胞分离快速，轻柔的手法。

<table><tbody><tr><td>(3-Aminopropyl) triethoxysilane (APTES)</td><td>Acros Organics</td><td>919-30-2</td><td>Used to make 2% APTES solution</td></tr><tr><td>Plasma Cleaner Pico</td><td>Diener</td><td>Model 1</td><td>Cleans surfaces and allows for bonding of PDMS to glass</td></tr><tr><td>d-Desthiobiotin (DSB)</td><td>Sigma</td><td>D20655</td><td>Used as the releasing mechanism in the cellular capture surface.&amp;nbsp;</td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>British Drug Houses (BDH)</td><td>BDH1115-1LP</td><td>Dissolves the DSB into solution</td></tr><tr><td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td><td>Thermo-Scientific</td><td>5g: 22980&lt;br/&gt; 25g: 22981</td><td>Activates carboxylic acids and allows binding of proteins to glass surface.</td></tr><tr><td>uncoated 8-well culture slide</td><td>BD Falcon</td><td>Case of 24: 354118&lt;br/&gt; Case of 96: 354108</td><td>Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments</td></tr><tr><td>Glass bottom 24-well plates</td><td>MatTek</td><td>P24G-0-13-F</td><td>Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments</td></tr><tr><td>Mercaptoethanol</td><td>Science Lab</td><td>60-24-2</td><td>Used to quench reaction between EDC and DSB</td></tr><tr><td>4-Morpholinoethanesulfonic acid hydrate&lt;br/&gt; (MES Hydrate 99%)</td><td>Fisher Scientific</td><td>AC172590250</td><td>Used to make 0.1 M MES Buffer for use in EDC reaction</td></tr><tr><td>Precision Oven</td><td>Thermo Scientific</td><td>11-475-153</td><td>Used in curing of PDMS and APTES layer.</td></tr><tr><td>Titramax 1000 Shaker</td><td>Heidolph</td><td>13-889-420</td><td>Used to ensure even distribution of APTES on surfaces.</td></tr><tr><td>1x Streptavidin 5 mg [e7105-5mg]</td><td>Proteo Chem</td><td>9013-20-1</td><td>Biotin-binding protein. May cause irritation.</td></tr><tr><td>5 cm Glass Dish</td><td>Fisher Scientific</td><td>08748A</td><td>Used in HUVEC studies as well as future profiling studies.</td></tr><tr><td>14 cm Petri Dish with Cover</td><td>Sigma-Aldrich</td><td>Z717231</td><td>Used to hold samples being functionalized and transport them.</td></tr><tr><td>MCF7-GFP cells</td><td>Cell Biolabs</td><td>AKR211</td><td>Stored in liquid nitrogen</td></tr><tr><td>RAW264.7 mouse macrophages</td><td>ATCC</td><td>TIB-71</td><td>Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen.</td></tr><tr><td>TrypLE</td><td>Life Technologies</td><td>12605036</td><td>Stored in 100 ml at room temperature</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium</td><td>Cell Media Facility at School of Chemical Sciences at UIUC</td><td>50003PC</td><td>Supplier: Corning</td></tr><tr><td>Nonessential amino acids</td><td>Cell Media Facility at School of Chemical Sciences at UIUC</td><td>25-025-CI</td><td>Already added into DMEM by facility. Supplier: Corning.</td></tr><tr><td>Cell scraper</td><td>Fisher Scientific</td><td>12-565-58</td><td>Small 23 cm 50 pack</td></tr><tr><td>Cell Dissociation Solution</td><td>Corning</td><td>MT-25-056CI</td><td>Used to lift cells non-enzymatically for the use in cell experiments</td></tr><tr><td>Hemacytometer</td><td>Hausser</td><td>02-671-54</td><td>Used to count cells for quantification of cell solutions and capture and release effectivity.</td></tr><tr><td>Biotin</td><td>Amresco</td><td>58-85-5</td><td>Used to release cells from surface.</td></tr><tr><td>HBSS</td><td>Created from Recipe</td><td>N/A</td><td>Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. Adapted from Cold Spring Harbor Protocols: In 500&amp;nbsp;ml, use 4&amp;nbsp;g NaCl, 0.2&amp;nbsp;g KCl, 0.0402&amp;nbsp;g Na&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O, 0.03&amp;nbsp;g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; and 0.5&amp;nbsp;g glucose. Add DI water to get to&amp;nbsp;500 ml, filter, and then refrigerate.</td></tr><tr><td>HLA-ABC Antibody</td><td>BioLegend</td><td>311402</td><td>Antibody used to capture MCF7gfp cells</td></tr><tr><td>hIgG Antibody</td><td>BioLegend</td><td>HP6017</td><td>Antibody used to capture MCF7gfp cells</td></tr><tr><td>MCF7 GFP cells</td><td>Cell Biolabs</td><td>AKR-211</td><td>Luminal Breast Cancer line that has been transfected with green fluorescent protein.</td></tr><tr><td>Assorted Conicals</td><td>Thermo-Scientific</td><td>15mL: 12-565-268</td><td>50/15 ml plastic conicals for storing solutions and aliquots.</td></tr><tr><td>Mini-Tube Rotators (End over End Mixer)</td><td>Fisher Scientific</td><td>05-450-127</td><td>Used to incubate antibody and mix other cellular solutions in order to mix</td></tr><tr><td>Axiovert 200M (Fluorescence Microscope)</td><td>Zeiss</td><td>N/A</td><td>Zeiss Axiovert 200 M inverted florescence microscope.</td></tr><tr><td>Zeba Desalting columns</td><td>Thermo-Scientific</td><td>PI-87770</td><td>Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf</td></tr><tr><td>EZ Link Sulfo NHS Low Weight Biotinylation Kit</td><td>Thermo- Scientific</td><td></td><td>Used to biotinylate antibodies to allow them to integrate with the capture surface</td></tr><tr><td>Plate Reader</td><td>BioTek</td><td>Synergy HTX Multimode Reader</td><td>Used to quantitatively measure fluorescent intensity in the titration experiments.</td></tr></tbody></table>

a method of targeted cell isolation via glass surface functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

使用该协议，我们表明细胞捕获（ 图3A）和MCF7GFP细胞的细胞释放（ 图3C）以及活细胞对照（ 图4）。我们量化的细胞捕获为60％和80％被释放（ 图3C）。当我们扩展这种方法的RAW 264.7巨噬细胞和MCF7GFP细胞的混合物，RAW巨噬细胞的50％被捕获（ 图3D）和RAW巨噬细胞有80％是释放用20mM生物素（ 图3B）。...

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A Method of Targeted Cell Isolation via Glass Surface Functionalization

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

通过玻璃表面功能靶细胞提取方法

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

a-method-targeted-cell-isolation-via-glass-surface

Research

JoVE Journal

Cancer Research

9.3K 次观看.  University of Illinois at Urbana-Champaign. This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process. 

视频: A Method of Targeted Cell Isolation via Glass Surface Functionalization

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

科研

癌症研究

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

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

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 ...

生物工程

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

分离和新型表面标记组合来标识肝祖亚群富集

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

发育生物学

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, such as nucleic acids and proteins. Single molecule FRET (sm-FRET) measurements on immobilized molecules permit long observations of the system -effectively until both dyes photobleach- resulting in time-traces that report on biomolecular dynamics with a broad range of timescales from milliseconds to minutes. To facilitate the acquisition of large number of traces for statistical analyses, the process must be automated and the sample environment should be tightly controlled over the entire measurement time (~12 hours). This is accomplished using an automated scanning confocal microscope that allows the interrogation of thousands of single molecules overnight, and a microfluidic cell that permits the controlled exchange of buffer, with restricted oxygen content and maintains a constant temperature throughout the entire measuring period. Here we show how to assemble the microfluidic device and how to activate its surface for DNA immobilization. Then we explain how to prepare a buffer to maximize the photostability and lifetime of the fluorophores. Finally, we show the steps involved in preparing the setup for the automated acquisition of time-resolved single molecule FRET traces of DNA molecules.

In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

表面固定生物分子的单分子荧光检测的自动化系统

Fluorescence Resonance Energy Transfer (FRET) microscopy has been widely used to study the structure and dynamics of molecules of biological interest, ...

生物学

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

分离和<em&gt;离体</emVδ1的&gt;企业文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT细胞，胸腺外的αβT细胞祖

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

免疫与感染

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

制备光学质量玻璃表面研究巨噬细胞融合

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

用于细菌筛选、选择和分离的可光降解水凝胶接口

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

用于分离多功能循环肿瘤细胞的临床微流控芯片平台

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

Source: Chen S. et al. <a href="https://www.jove.com/video/56394" target="_blank">Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.</a> J. Vis. Exp. (2018)
In this video, we demonstrate the isolation of EpCAM-expressing epithelial cancer cells from peripheral blood samples. The technique involves the use of a medical-grade wire functionalized with EpCAM&#160;antibodies to capture the target cells. The captured cells are then subsequently extracted into a buffer solution.

功能化基于线的靶细胞分离:一种从加标外周血中分离癌细胞的离体技术

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)
In this ...