Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

包被的玻璃盖玻片1.准备二氧化钛<ol><li>号码使用金刚石划片盖玻片。这不仅有助于跟踪每个盖玻片的同时也确保了样品的正确的一面朝上。清洁的盖玻片，第一下运行DDH 2 O的，然后通过浸入它们食人鱼溶液（H 2 SO 4 :H 2 O 2 = 4：1）。 10分钟后，彻底冲洗盖玻片，8次双蒸2 O.在干燥N 2流盖玻片。 </li><li>在射频（RF）溅射系统设置的 TiO 2靶。附加的盖玻片上使用聚酰亚胺胶带将溅射设备的一个样品架。将样本保持在溅射室中。疏散室，直到压力达到2.0×10 -4帕。 </li><li>引入Ar气到腔室内，并设置在沉积压力为4.0毫乇。当保持闸板关闭时，逐渐增加RF功率至70瓦</li><li>打开快门和溅射15分钟，得到一个薄膜的厚度为120-150毫微米（ 图1：步骤A）。膜的生长速率需要被衍生为每个机。 </li></ol> 2.表面涂层与细胞排斥膜<ol><li>通过用O 2等离子体处理样品，以下由等离子体反应器的制造商提供的说明书亲水在TiO 2表面上。我们治疗的样品进行5分钟，在200瓦与O 2流量以100sccm。沉浸在样品中DDH 2 O的，并确认表面超亲水性。表面干燥彻底在N 2流。 </li><li>制备1mM的十八烷基三氯硅烷（OTS）溶液中加入39.6微升的OTS 100毫升甲苯。浸在1小时在室温下的溶液中的样品。进行此步骤内的N 2 -filled手套袋中（ 图1：步骤B）。 </li><li>要除去物理吸附的分子，sonicatE中的样品中的甲苯，丙酮，乙醇和DDH 2 O的，每次5分钟，在该顺序。冲洗样品四次新鲜的DDH 2 O的和干燥在N 2流的表面。表面应是疏水为100-110°的接触角。 </li></ol> 3. 前现场表面图案<ol><li>工作在层流罩，得出一些划痕，表面上的钻石划线。这些标记有助于跟踪处理过的区域，也使显微镜聚焦。通过浸渍在70％乙醇的样品5分钟消毒OTS涂覆的 TiO 2。然后用清水冲洗样品两次消毒的双蒸2 O. </li><li>放置在一个35毫米的培养皿的样品，并加2ml的PC12生长培养基（参见步骤4.2）。孵育超过3小时，在CO 2培养箱中（37℃）。这个程序的目的是让血清白蛋白吸收在表面上。吸附白蛋白抑制其他prote随后的吸附插件和细胞 （图1：步骤C）。 </li><li>在等待时，设置了倒置荧光显微镜。 <ol><li>打开弧光灯，插入UV滤光立方体，和物镜设置为20X。 </li><li>测量光强度 I（W厘米-2）用紫外线强度计，并计算辐照时间 t（秒）为一个在剂量d的（以J厘米-2）为：T = D / I。 例如，照射在为200J厘米-2的剂量使用的600毫瓦cm -2的光源，333秒的照射是必需的。 </li><li>使用一个阶段微米和关闭场光阑设置要被照射的区域的尺寸，例如，200微米。 </li></ol></li><li>在3小时温育后，补充用200μl的3.0毫克毫升-1 IV型胶原的培养基（COL-IV;最终浓度300微克毫升-1）。 </li><li>转移35毫米的菜显微镜阶段。发现划痕米柜，聚焦显微镜到样品表面上，并且照射UV光以为200J cm -2的（ 图1：步骤D）的剂量。紫外线照射的面积可以通过调整视场光阑的开口，或通过与仲裁几何形状的金属掩模代替场光阑被改变。 </li><li>替换为新鲜生长培养基的培养基（无COL-IV）中，并将样品放回到培养箱。 </li></ol> 4.细胞培养<ol><li> PC12细胞常规培养进行在胶原包被的塑料盘。 <ol><li>涂覆60mm的组织培养皿山口-Ⅳ，第一润湿表面与DDH 2 O的，吸所有双蒸2 O. </li><li>用双蒸2 O稀释原液（3毫克毫升-1）10X准备300微克毫升-1 COL-IV </li><li>加入200μl的300微克毫升-1每60毫米的菜COL-IV。让该溶液通过表面扩散。 </li><li>干吨他菜在层流罩约1小时。 </li><li>用4ml的Dulbecco磷酸盐缓冲盐水（D-PBS）的漂清表面的两倍。如果不立 ​​即使用的涂层菜，用4mL双蒸2 O.的涮菜一次吸气DDH 2 O的后，保存在孵化器的菜。尽量不要把它存放超过两个星期。 </li></ol></li><li> PC12细胞生长在该组成的生长培养基：Dulbecco改良的Eagle培养基（低葡萄糖）+ 10％胎牛血清+ 5％马血清+ 1％青霉素 - 链霉素溶液。孵育细胞在 CO 2培养箱中（37℃，5％的CO 2），并更换一半培养基每隔一天。传代细胞才达到汇合。 <ol><li>吸出生长培养基，并加入2毫升PBS +（D-PBS + 10毫克毫升-1牛血清白蛋白（BSA）+ 10mM EDTA的）预热至37℃。孵育5分钟，在37℃。轻轻敲击菜分离所有CELLS。 </li><li>收集细胞在15ml锥形管中。冲洗用3毫升新鲜的D-PBS，预热至37℃的菜。 </li><li>离心反应管，在150×g下4分钟。 </li><li>吸出上清液，并添加1毫升生长培养基。 </li><li>对于常规培养，分裂细胞1：3至1：5。 </li></ol></li><li>板块细胞对紫外线改性的OTS / TiO 2的样品，计数细胞密度，并在35毫米培养皿（ 图1：步骤E）添加3.0×10 5个细胞 。孵育在加湿培养箱的培养皿（37℃，5％CO 2）1-2天。既幼稚和神经生长因子（NGF）-differentiated PC12细胞可用于构图实验。对于NGF分化的7S-NGF 100纳克毫升-1电镀细胞到样品之前加入到生长培养基中数天。 NGF分化似乎增加了PC12细胞的粘附性和使处理更容易，尤其是在原位图案化Éxperiments。 </li></ol> 5. 原位表面图案<ol><li>经过1-2天的培养上易地修饰表面，确认细胞附着并只在紫外线照射区域增长。设置的显微镜，如步骤3.4中所述。 </li><li>将样品转移到含有生长培养基的新35毫米组织培养皿，100纳克毫升-1 NGF（在使用NGF分化的细胞的情况下），和100μg毫升-1 COL-IV。 </li><li>放在显微镜阶段的菜。找到相应的位置，并且照射UV光以为200J cm -2的（ 图1：步骤F，G）的 ​​剂量。新创建的许可区域表示与图1中以星号：步骤G </li><li>尽量在30分钟内完成一个样品的处理。照射后，将样品转移回未经COL-IV。 </li><li>携带与铜的菜时，要特别小心ltured细胞和从盘传送样品盘，以防止拆卸图案的细胞。 </li></ol><img alt="图1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> 图中，整个过程1示意图。详见说明书。 <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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The authors have nothing to disclose.

在我们目前的协议， 二氧化钛膜通过射频磁控溅射形成。我们是有利于沉积的这种方法，因为它允许我们可重复使用亚纳米粗糙度调制成光催化二氧化钛薄膜。虽然溅射淀积过程是熟悉的材料科学家和电子工程师，它可能不是很访问生物学家。在这种情况下，旋涂的TiO 2膜 。将另外的选择23。在该方法中， 二氧化钛纳米颗粒溶解在溶剂中通过离心力铺展?...

半导体光刻工艺及其衍生物-诸如光刻1,2，电子束光刻3-6，和微接触印刷7-10 -现在已成为一个既定的工具在细胞生物学生长的活细胞在限定位置和几何形状。图案形成方法依赖于使用微制造基底的，由微岛细胞容许涂料在非允许的背景。例如基底用作模板图案的细胞。这些技术为我们提供了新的方法来设计的细胞和它们的功能在一个单和多细胞水平上，以提取细胞的固有特性，并增加细胞为基础的药物筛选11的吞吐量。 在细胞图案化的自由度度会大大增加，如果模板图案几何可以改变在原位 ， 即 ，当细胞是上一次一培养urface。常规方法的图案形成，不能直接应用于这里，因为它们处理样品中的气氛或真空。因此各 ​​种新型的表面改性技术已被提出，它是基于，例如，在光反应性化合物12,13或激光烧蚀5,14，仅举几例。所提出的方法已经由Robertus 等人已很好地审查 。15，以及最近由Choi 等人的 16和中西17。 在这里，在这篇文章中，我们描述了一种新的原位表面修饰，这需要对二氧化钛的优势有机分子的光催化分解（ 二氧化钛）表面18,19协议。在该方法中，TiO 2膜被插入在玻璃基板和接口单元中的有机薄膜之间，且有机薄膜通过局部地照射紫外线（UV）的分解原位光到感兴趣的区域（λ&lt;388纳米）。我们表明，新的协议可以用于创建细胞外基质蛋白和活细胞双方易地和原位微图案。 二氧化钛是生物相容的，化学稳定的，且光学透明的，特征，其中使得它友好引入在细胞培养实验。该协议提供了一个材料科学为基础的替代修改细胞培养支架的细胞培养环境。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

吸附的二氧化钛（TiO 2的）的表面上的有机污染物可以通过光催化下的紫外线（UV）光分解。在这里，我们描述了一种新的协议采用二氧化钛光催化局部改变衬底表面的细胞亲和力。在这个实验中，一个薄的TiO 2膜为溅射镀膜在玻璃盖玻片，并在TiO 2表面随后改性以从十八烷基三氯硅烷（OTS）衍生的有机硅烷单层，其抑制细胞粘附。将样品浸在细胞培养基中，和聚焦UV光照射至一个八角形区域。当神经元细胞系PC12细胞铺在样本，细胞粘附只在紫外线照射的区域。进一步的研究表明这种表面改性也可以在原位进行，也就是说，即使当细胞生长在衬底上。适当的修改表面的所需的细胞外矩阵Protein胶原存在于在紫外线照射时的介质。这里提出的技术可以潜在地在图案形成多种细胞类型可以采用用于构建共培养系统或任意地操纵细胞培养下。

图2A示出了溅射沉积的 TiO 2膜的横截面扫描电子显微镜（SEM）图像。从观察，膜的厚度估计为大约150纳米。引人注目这里是所沉积的TiO 2膜的平整度。通过原子力显微镜（AFM）进一步分析显示，在表面的根均方（RMS）粗糙度为0.2纳米（ 图2B）。 当TiO 2的表面被改性以OTS，然后浸渍在含有血清的生长培养基中，血清白蛋白吸附到...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

光图案蛋白质和细胞中的水环境中使用二氧化钛<sub&gt; 2</sub&gt;光催化

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Research

JoVE Journal

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

7.8K 次观看.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

视频: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

负染色的EM可视化蛋白质和大分子复合物：从电网的制备图像采集

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

一个工具包，使水环境中的烃类转化

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

利用双水相系统的细胞共培养图案

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

用作酶反应器的水滴及其电磁驱动

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...