我们承认奥斯汀Cyphersmith和Mayandi Sivaguru（卡尔R. Woese研究所基因组生物学，伊利诺伊大学厄巴纳 - 香槟分校），与显微镜援助和在显微镜核心慷慨地容纳屏和视频捕捉。

作者宣称，他们没有竞争的经济利益。

在我们的实验中，我们已经发现，最常见的故障都涉及到制造的阵列的质量和差的特征在于在感兴趣的生物系统响应。我们建议读者参考表1在细胞微阵列实验常见失效模式和相关的故障排除步骤。特别是关于阵列的质量，我们推荐以下。确认排列程序，参数，以及使用荧光标记的分子，例如若丹明共轭葡聚糖缓冲器的技术质量和鲁棒性。彻底清洁针之前或按照制造商的说明书排列，并进一步在视觉上后检查销渠道使用光学显微镜是明确的碎片。使用通用的蛋白质染色或免疫标记确认阵列生物分子的保留。需要注意的是低于70 kDa的分子量的生物分子经常不保留在水凝胶23， </<html> SUP&gt; 31。使用多种细胞类型验证阵列的生物分子的细胞的功能。请注意，只有贴壁细胞与阵列兼容;另外，粘附到阵列是依赖于两个小区特定属性（ 例如 ，整联表达谱），并将选定ECM蛋白。 由于篇幅有限，我们这里不提供阵列设计，布局和制造广泛的治疗和读者参考以前的作品23，25。我们一般使用10-20独特的生物分子的条件（ 即 ，5-10个/条件）组成的100点的子阵列（150微米的光斑直径，450微米的中心-至-中心距离）。子阵列的一个阵列的数量取决于所感兴趣的生物分子的条件的数量，这可以舒适地按比例放大到1,280上的一个25×75毫米的显微镜载玻片（〜6400点的64子阵列）外部参照"&gt; 25,31以上参数将取决于所感兴趣的图案尺寸进一步变化;能够从75产生图案的销- 450微米都是现成的。 阵列实验最好使用其他文化形式的利息高得分排列的情况下，法显装置和生物模型系统验证补充。具体而言，我们建议使用批量培养符合标准的分子生物学技术（ 例如 ，定量RT-PCR，免疫印迹）或标准TFM一起进一步验证的选择摆着条件的影响。遗传操作在一个适当的生物模型系统还可以用来确认在阵列观察效果感兴趣的因子（ 例如 ，敲除或过表达）。 在体内动物模型表示验证的另一种手段和最近使用的，例如，以确认在半乳凝素-3和半乳凝素8的中心作用肺癌转移的利基，如通过细胞芯片31，49初步确定。 其他一些方法已被用来探测细胞功能的微环境调控，包括各种二维微加工系统18，50，51，52，53，54，55和三维设计的生物材料系统56，57，58 ，59，60，61。与其他方法相比，这里描述的细胞微阵列平台的特别的优点包括:（1）的吞吐量达到数百或数千的因素的不同组合，可实现的相互作用效应的分析; （2）访问，自动成像和分析; （3）与排列因素控制介绍生物化学和生物物理读数的整合; （4），以改变基底材料特性的能力;和（5）含量高的细胞命运和功能的单细胞分析。 综上所述，随着TFM细胞芯片的可调谐基板刚性基材的结合使双方生物化学和生物物理线索的深入刻画。如这里介绍的，该平台是一般化并可以容易地应用于各种贴壁细胞类型和组织的上下文向细胞分化和机械传导的组合微环境调节的改进的理解。

细胞和周围微环境因素之间的相互作用介导了大量的各种整个发育，稳态和疾病的发病机制1，2，3，4的生物过程。这些微环境相互作用包括通过配体 - 受体结合递送的可溶性因子的细胞，细胞 - 基质结合，和细胞 - 细胞相互作用。除了上述的生化考虑，生物物理参数，如底物的机械性能（ 例如 ，杨氏模量，孔隙率）和电池的形状，和相关的下游机械传导日益得到认可作为细胞分化5，6，7，8的关键介质， 9 <sup&gt; 10。从这些微环境相互作用产生的信号作为输入，基因网络和信号通路。此外，这些细胞的固有组分也通过分泌的因子和基质降解酶，完成细胞内在遗传计划和细胞外微环境因素5，11，12之间的复合物共同监管环路反馈提供给微环境。 使用用于微环境因素的控制介绍工程系统的范围内不同的上下文13，14，15的已被证明是有用的。特别是微制造的系统已通过小型化13促进蛋白质和细胞以及高度并行分析的精确的空间图案化<sup类="外部参照"&gt; 16，17，18，19，20，21，22。细胞微阵列表示一个这样的微加工系统，其中的生物分子的组合是接触印刷到弹性聚丙烯酰胺水凝胶基底23，24，25。细胞粘接性成分（即基质蛋白）的加入使芯片上的持续细胞粘附和文化，这是经常其次是通过免疫组化和荧光记者下游分析。细胞芯片已经富有成效针对实现肝细胞表型的23，26，神经前体分化27 mammar的改进认识ý祖命运决定28，胚胎干细胞的维持/分化23，29，30，肺癌转移31，以及在黑色素瘤32的治疗反应。我们最近证明了使用细胞微阵列的用于限定在定形内胚层规范33，肝脏祖细胞分化34，35，和肺肿瘤细胞对药物的反应36外基质（ECM）蛋白的组合物的作用。在这些作品中，我们把重点放在扩大阵列平台的组合能力和探索细胞内在信号与细胞外基质成分及生物力学的交叉点。此外，我们在这个阵列平台上实现生物物理读数能力提供定量CHARAC细胞收缩的分化terize作用处理35。要做到这一点，我们综合牵引力显微镜（TFM）与细胞芯片，使细胞产生牵引的高通量评估。 TFM是测量细胞产生的牵引力一种广泛使用的方法，并提供了有关单细胞和组织水平的功能与组合物和局部微环境37，38，39，40的生物力学的协调显著见解。因此，TFM与细胞微阵列结合提供了一个高通量系统，用于测量键，生理学相关的生物物理参数。 这里描述的细胞微阵列平台由四部分组成:聚丙烯酰胺基板，阵列，细胞培养和测定的读出的制造，以及数据分析的制造。看到图1为前三个实验部分的示意性概要;参见图2的最后一节的重点放在免疫荧光分析数据的概要总结。为了适应在细胞微阵列平台来生物力学细胞-基底相互作用的研究中，我们使用可调谐的杨氏模量，但相似的孔隙率的聚丙烯酰胺底物，每Wen 等。 41。以使由它们的底物对细胞施加的力的TFM测量，我们实现除了厚的玻璃显微镜载片经常被其它基团使用的玻璃底培养皿的格式。因此，这种细胞芯片平台能够细胞分化通过对显微镜载玻片和细胞产生的力通过免疫荧光TFM单独的玻璃底菜并行测量。我们也应用了一些改进通常与电池芯片使用的分析方法。 SpecificalLY，代替整体岛强度的参数的Z得分，我们测量的单细胞的强度和以占非正常分布和更准确地描述细胞行为申请位数正常化。我们相信，这些改进提供了走向其生物化学和生物物理线索发挥显著，交叉作用的生物学过程的调查特别有用。此外，我们的分析改进使细胞芯片的一系列对这些单细胞和群体水平的行为发散细胞功能研究中的应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;m syringe filter</td>
			<td>Pall Corporation</td>
			<td>4433</td>
			<td>Match with appropriately-sized Luer lock plastic syringes.</td>
		</tr>
		<tr>
			<td>100&#215; penicillin&#8211;streptomycin solution</td>
			<td>Fisher Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>22&#215;60 mm coverglasses</td>
			<td>Electron Microscope Sciences</td>
			<td>63765</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(trimethoxysilyl)propyl methacrylate (3-TPM)</td>
			<td>Sigma-Aldrich</td>
			<td>440159</td>
			<td>Store under inert gas per manufacturer&#39;s instructions. Exposure of 3-TPM to air could compromise silanization of glass substrates. CAUTION: 3-TPM is a combustible liquid. Keep away from heat, sparks, open flames, and hot surfaces and use only in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS)</td>
			<td>Sigma-Aldrich</td>
			<td>C3023</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm glass-bottom Petri dishes</td>
			<td>Cell E&#38;G</td>
			<td>GBD00002-200</td>
			<td>13 mm well consisting of #1.5 coverglass. Enables TFM and live-cell imaging.</td>
		</tr>
		<tr>
			<td>384-well polypropylene V-bottom microplate, non-sterile</td>
			<td>USA Scientific</td>
			<td>1823-8400</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well polystyrene microplates</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td>35 mm glass-bottom Petri dishes fit into wells of microplate, easying array fabrication.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179973</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A3553</td>
			<td>CAUTION: Exposure to acrylamide can result in acute toxicity and irritation. Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail</td>
			<td>EMD Millipore</td>
			<td>08-115MI</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen III, human</td>
			<td>EMD Millipore</td>
			<td>CC054</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV, human</td>
			<td>EMD Millipore</td>
			<td>CC076</td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinker, 365 nm</td>
			<td>UVP</td>
			<td>CL-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, rhodamine B-conjugated, 70 kDa</td>
			<td>ThermoFisher Scientific</td>
			<td>D1841</td>
			<td>Used as a marker for array location.</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (PBS)</td>
			<td>Fisher Scientific (HyClone)</td>
			<td>SH3001302</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-recombinant DLL1, mouse</td>
			<td>R&#38;D Systems</td>
			<td>5026-DL-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-recombinant DLL4, mouse</td>
			<td>AdipoGen</td>
			<td>AG-40A-0145-C050</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-recombinant JAG1, rat</td>
			<td>R&#38;D Systems</td>
			<td>599-JG-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin, human</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope, inverted</td>
			<td>Zeiss</td>
			<td>Axiovert 200M</td>
			<td>Ensure microscope is equipped with a robotic stage for both automated fluorescent imaging and TFM. Environmental control (i.e., 37 &#176;C and 5% CO2) is highly advisable for TFM.</td>
		</tr>
		<tr>
			<td>Fluoromount G with DAPI</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>695092</td>
			<td>CAUTION: Acetic acid is flammable and corrosive. Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr>
			<td>Irgacure 2959</td>
			<td>BASF Corporation</td>
			<td>55047962</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin, mouse</td>
			<td>EMD Millipore</td>
			<td>CC095</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>179957</td>
			<td></td>
		</tr>
		<tr>
			<td>Microarray scanner</td>
			<td>GenePix</td>
			<td>4000B</td>
			<td>Fluorophores must be Cy3- or Cy5-compatible.</td>
		</tr>
		<tr>
			<td>Microarrayer</td>
			<td>Digilab</td>
			<td>OmniGrid Micro</td>
			<td>Other microarrayerse of similar or greater capability can readily be substituted.</td>
		</tr>
		<tr>
			<td>Microscope slides, 25&#215;75 mm</td>
			<td>Sigma-Aldrich</td>
			<td>CLS294775X25</td>
			<td>~0.9&#8211;1.1 mm thickness.</td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebisacrylamide (bisacrylamide)</td>
			<td>Sigma-Aldrich</td>
			<td>M7279</td>
			<td>CAUTION: Exposure to acrylamide can result in acute toxicity and irritation. Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA), 16% v/v</td>
			<td>Electron Microscope Sciences</td>
			<td>RT15710</td>
			<td>Prepare PFA fresh (do not store) for optimal fixation. CAUTION: Exposure to PFA can result in acute toxicity and can also irritate or corrode skin on contact. Wear protective gloves, clothing, and eye protection and use only in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Protein A/G, recombinant</td>
			<td>ThermoFisher Scientific</td>
			<td>21186</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex drying tray, 2000 ml</td>
			<td>Fisher Scientific</td>
			<td>15-242B&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular 4-chambered culture dish</td>
			<td>Fisher Scientific (Nunc)</td>
			<td>12-565-495</td>
			<td>For cell culture on arrayed microscope slides.</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>415413</td>
			<td>CAUTION: NaOH is highly caustic and can cause severe skin burns and eye damage. Wear protective gloves, clothing, and eye protection.</td>
		</tr>
		<tr>
			<td>Stealth pin for arraying</td>
			<td>ArrayIt</td>
			<td>SMP3</td>
			<td>Clean pins after each array run using the instructions of the manufacturer. Produces 150 micron domains; purchase other pin sizes (75&#8211;450 microns) as suited to your particular application.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput cell microarray platform for correlative analysis of cell differentiation and traction forces

微制造蜂窝微阵列，其上的弹性的水凝胶表面由生物分子的接触印刷的组合，用于测量排列生化信号对细胞分化的影响提供一个紧密控制的，高通量的设计系统。采用细胞芯片最近作出的努力证明他们的组合研究中，许多微环境因素的并行呈现效用。然而，这些努力主要集中在调查生化线索对细胞反应的作用。在这里，我们提出了一个细胞芯片平台具有可调的材料特性为牵引力显微镜评估由免疫和生物力学的细胞 - 基底相互作用的两个细胞的分化。要做到这一点，我们已开发利用不同的杨氏模量在任显微镜载玻片或玻璃底培养皿中制成的聚丙烯酰胺水凝胶两种不同格式。我们提供BESŧ实践和故障排除的微阵列对这些水凝胶基质制造，芯片上的后续细胞培养和采集数据。该平台非常适合用在生物过程的调查对其中两个生化（ 例如 ，细胞外基质组合物）和生物物理（ 例如 ，基板的刚度）的线索可能发挥显著，相交的作用。

使用此平台，我们研究在肝脏祖34,35的命运规范生物化学和生物物理线索的作用。 A / G蛋白偶联Notch配体显示出改善的保持和集群中的聚丙烯酰胺水凝胶（ 图3A），并还能够驱动肝祖细胞分化走向胆管细胞命运（ 图3B）。使用单细胞分析，我们量化的Notch配体的ECM蛋白胶原蛋白I，胶原蛋白III，胶原蛋白IV，纤连蛋白，和层粘连蛋白（ 图3C）的反应，发现肝脏祖细胞的配体的反应也取决于ECM上下文。最后，我们利用shRNA的敲除产生肝祖没有配体DLL1和JAG1。向排列Notch配体的反应向变取决于公关任一配体的esence，确认该响应的细胞外配体也是细胞内在配体表达（ 图3D）的功能。此外，我们观察到双阳性的一个独特的亚群（ALB + / OPN +）细胞中的DLL1击倒（ 图3D）。总之，这些代表性的结果表明:（1）阵列格式的组合能力，例示由多个排列ECM蛋白和Notch配体与个别的配位体的击倒的配对; （2）不仅功能排列ECM蛋白，但也可通过蛋白A / G介导的偶联排列的细胞间的配体; （3）我们的单细胞分析和辨别独特的亚群的能力的实现。 我们还观察到肝祖细胞的分化取决于衬底的刚度和ECM组合物（ 图4A上都</str<html>翁&gt;），特别是发现IV型胶原是软，硬衬底支持的分化，而纤连蛋白只支持刚性基板（ 图4B）的分化。 TFM测量的代表热图表明，在对IV型胶原低的衬底的刚度持续牵引应力促进分化成胆管细胞（ 图4C），通过平均根均方值（ 图4D）证实这一发现。总之，这些代表性的结果表明:（1）TFM的成功整合与细胞微阵列上带有可调刚性基板来评估两个细胞表型和牵引应力; （2）同时与基质组合物和基板的刚度肝祖细胞命运的协调;及（3）在细胞微阵列我们的TFM分析和典型的牵引应力分布的执行。 Ë1"SRC ="/文件/ ftp_upload / 55362 / 55362fig1.jpg"/&gt; 图1:概述示意图，显示前三的实验部分。在第一节中，玻璃基板清洗和硅烷化，以促进聚丙烯酰胺水凝胶的制备。在第2节中，感兴趣的生物分子的组合在384孔微孔板源制备。然后，机器人点样仪装有干净引脚，源微孔板和聚丙烯酰胺水凝胶和初始化，制造的水凝胶阵列。在第3节，细胞接种到阵列域和允许粘附，进行兴趣培养协议之后。在终点，细胞是固定的免疫细胞化学/免疫荧光分析或使用TFM。比例尺是75微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55362/55362fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ="1"&gt; - 页面内<img alt="图2" src="/files/ftp_upload/55362/55362fig2.jpg" /> 图2:处理和阵列免疫荧光数据分析。 （A），瓷砖，复合32位RGB图像首先装箱，然后分割成单独的8位通道。使用排列的荧光标记物和细胞岛的组合，阵列的三个角被确定，以允许自动定向和阵列网格。 （B）中的单细胞数据是为输入阵列的每一个通道产生的。为了解释实验漂移，分位数归一化是通过生物复制施加，产生在所有重复单个共享分布。分位数归一化的数据随后被绘制并经由合奏测量计算解释（ 例如 ，细胞/岛，平均强度，正面为一标签百分比细胞）或单细胞分布的直接分析。M /文件/ ftp_upload / 55362 / 55362fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/55362/55362fig3.jpg" /> 图3:Notch配体介导介绍祖肝脏分化。 （A）的Fc重组Notch配体铁血-1（JAG1）和Delta样1（DLL1）展出时蛋白A / G摆着改进保留和集群。比例尺为50微米。 （二）肝祖细胞在与Notch配体呈现分化成胆管细胞。 4&#39;，6-二脒基-2-苯基吲哚（DAPI）是一种核标签，白蛋白（ALB）是肝细胞标志物，以及骨桥蛋白（OPN）是胆管细胞标记。比例尺为150微米。 （C）细胞百分比阳性OPN上的ECM蛋白的Notch配体JAG1，DLL1和Delta样4（DLL4）的量化胶原蛋白I，Collagen III，IV型胶原，纤连蛋白，层粘连蛋白。学生t -tests分别对每个ECM蛋白中的每个排列Notch配体与P值为P &lt;0.05（*）表示对对照IgG进行。 （D）成像ALB和OPN对胶原III细胞呈现与Notch配体JAG1，DLL1和DLL4流式细胞仪。无缺口肝祖细胞配体DLL1和JAG1（ 即 shDll1和shJag1）用shRNA的敲除产生。在（C）的数据表示为平均值±SEM这个数字已从Kaylan 等改性。 34。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55362/55362fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55362/55362fig4.jpg" /> 图4:基质成分和基质刚度首席运营官rdinate肝祖分化。 （一）肝祖分化为胆管细胞是依赖于这两个ECM成分和基质刚度。 DAPI是核标签，ALB是肝细胞标记，OPN是胆管细胞标志物。 （B）中的正面为OPN细胞百分比上的杨氏模量的基片30千帕，13千帕，并在4kPa用于胶原I（C1）的，IV型胶原（C 4），纤连蛋白（FN），以及所有的双向组合的定量这些细胞外基质蛋白。 （C）细胞牵引压力取决于两个基底刚度和细胞外基质成分上。 （D）上的杨氏模量的基片30千帕和4千帕为胶原蛋白I（C1），IV型胶原（C 4），纤连蛋白（FN），这些的所有双向组合牵引应力的根均方值的量化ECM的蛋白质。在（B）和（D），数据以平均值±SEM和学生t -tests分别针对30千帕执行用于与P值每个的ECM组合为P &lt;0.05（*），P &lt;0.01（**）表示，和P &lt;0.001（***）。比例尺是50微米。这个数字已经从Kourouklis 等进行修改。 35。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55362/55362fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="1.2in"> 部分 </td><td fo:width="1.5in"> 问题 </td><td fo:width="1.5in"> 可能原因 </td><td> 解 </td></tr><tr><td rowspan="3"> 1.制造聚丙烯酰胺基质。 </td><td>玻璃罩不能从水凝胶中移除。 </td><td>过聚合。 </td><td>减少聚合时间&lt;10分钟（4瓦/米2）。检查UV crossli北部重点经济区产量预期范围之内。 </td></tr><tr><td>可怜的聚丙烯酰胺水凝胶的聚合。 </td><td> Underpolymerization。 </td><td>增加聚合时间&gt; 10分钟（4瓦/米2）。检查UV交联剂产量预期范围之内。 </td></tr><tr><td>聚丙烯酰胺水凝胶去除玻璃罩后损坏。 </td><td>软聚丙烯酰胺水凝胶是容易损坏。 </td><td>我们观察减少为最柔软的特别（ 即 4千帕）的水凝胶的水凝胶制造的成品率（〜50％）。轻拿轻放的水凝胶，增加的出发人数达到期望的收益率。 </td></tr><tr><td rowspan="2"> 2.制作阵列。 </td><td rowspan="2">可怜的或不一致的斑点形态。 </td><td>加湿器不一致功能。 </td><td>检查加湿器和贯穿每个印数流变仪的功能，并保持65％RH。 </td></tr><tr><td>卡在打印头堵塞或针GED。 </td><td>清洁打印头，允许自由运动的针。干净彻底针或前每个打印运行之后，从销渠道除去聚集。 </td></tr><tr><td rowspan="8"> 3.细胞培养和试验执行。 </td><td rowspan="2">细胞脱落或死亡初始附件后阵列。 </td><td>播和过度增生。 </td><td>降低了初始接种密度和时间。使用数组文化中"维修"或"分化"媒体以减少细胞增殖。 </td></tr><tr><td>从有毒的水凝胶丙烯酰胺单体的释放。 </td><td>水凝胶浸泡在蒸馏水中2 O，至少3天，以允许丙烯酰胺单体的扩散/释放和减少细胞毒性。 </td></tr><tr><td rowspan="4">细胞不重视阵列。 </td><td> Underseeding。 </td><td>增加初始接种密度和时间。使用更强烈粘附的细胞类型。 </td></tr><tr><td>贫困沉积矩阵或生物分子的条件。 </td><td>颗粒和聚集体的清洁销，确认印刷参数，并且评估荧光标记， 例如 ，若丹明共轭葡聚糖斑点。 </td></tr><tr><td>细胞 - 基质相互作用的特异性。 </td><td>不同的细胞类型专门坚持一些而不是其他细胞外基质蛋白。测试多个不同的ECM蛋白与你的细胞。 </td></tr><tr><td>加工后的次优阵列存储。 </td><td>我们建议在65％RH和室温下存放过夜捏造阵列，部分冻结期间避免阶段的变化。细胞粘附是既湿度，温度和存储时间敏感;确保这些参数都是一致的/你的实验进行了优化。 </td></tr><tr><td rowspan="2">细胞培养过程中从玻璃基板的水凝胶的脱离。 </td><td>可怜的滑动清洗和硅烷化。 </td><td>幻灯片清洗更换工作方案和硅烷化。 </td></tr><tr><td> Overdehydrated水凝胶。 </td><td>不离开水凝胶在热板上脱水长于15-30分钟。 </td></tr><tr><td> 4.分析数据。 </td><td>重复点和幻灯片之间的高变异性。 </td><td>变异阵列制造。 </td><td>检查引脚和打印头清洁。确认加湿器的功能。可视化和使用荧光标记量化现货和阵列质量。存储阵列如上建议。 </td></tr></tbody></table> 表1:故障排除。

Watch this Scientific Journal Video about A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces at JoVE.com

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

细胞分化是由微环境因素的影响，包括基质组合物和基材的材料性质的宿主调节。我们在这里描述利用细胞微阵列与牵引力显微镜一起同时评估细胞分化和生物力学细胞 - 基底相互作用的微环境上下文的功能的技术。

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

a-high-throughput-cell-microarray-platform-for-correlative-analysis

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K 次观看.  University of Illinois at Urbana–Champaign. 细胞分化是由微环境因素的影响，包括基质组合物和基材的材料性质的宿主调节。我们在这里描述利用细胞微阵列与牵引力显微镜一起同时评估细胞分化和生物力学细胞 - 基底相互作用的微环境上下文的功能的技术。 

视频: A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

科研

生物工程

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

高吞吐量的单细胞和多细胞微胶囊

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

一种多功能自动化平台微尺度细胞刺激实验

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

细胞分化和牵引力的相关分析高通量细胞芯片平台

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高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

聚合物芯片高通量发现的生物材料

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高吞吐量的单细胞和多细胞微胶囊

相关显微镜的动力相互作用的三维结构分析

一种多功能自动化平台微尺度细胞刺激实验

在细胞和组织力学生物学应用一种新型的拉伸平台

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

高通量单细胞的分离和文化的微流体平台

细胞分化和牵引力的相关分析高通量细胞芯片平台

摘要

探索更多视频

此视频中的章节

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidig​​m公司动态ArrayTM国际金融中心

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

聚合物芯片高通量发现的生物材料

利用微流控平台的高通量蛋白质表达发生器

高吞吐量的单细胞和多细胞微胶囊

相关显微镜的动力相互作用的三维结构分析

一种多功能自动化平台微尺度细胞刺激实验

在细胞和组织力学生物学应用一种新型的拉伸平台

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

高通量单细胞的分离和文化的微流体平台

高通量微RNA分析:优化复纳升秤定量RT - PCR Fluidigm公司动态ArrayTM国际金融中心