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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.</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 
			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 
			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 
			(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 5mg 
			[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 100mL at room temperature</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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>10% fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>03-600-511</td>
			<td>Stored in 500mL at &#60; -10&#8304;C</td>
		</tr>
		<tr>
			<td>1% Penicillin&#8211;Streptomycin</td>
			<td>Life Sciences Storeroom at UIUC</td>
			<td>17602e</td>
			<td>Supplier: VWR 
			Stored in 100 ml at 4&#8304;C</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>12-565-58</td>
			<td>Small 23cm 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 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 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）。...

Watch this Scientific Journal Video about A Method of Targeted Cell Isolation via Glass Surface Functionalization at JoVE.com

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

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

肿瘤微环境的模仿:产生富集的细胞群及侦查间通讯的简单方法

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

科研

癌症研究

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

光动力疗法的抗癌功效肺癌靶向纳米粒子

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

复杂核型细胞周期捕获细胞的生成与分离

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

表面增强共振拉曼散射纳米探针比比法检测微卵巢癌的意义上

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

从3维球体培养中分离出类似干细胞的细胞

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

使用 HiBiT CRISPR 细胞系对靶向蛋白质降解化合物进行高通量细胞分析

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

研究利普托梅宁格疾病直接靶向治疗的穆林·奥马亚异种格拉夫特模型

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...