作者想感谢慷慨提供H1的干细胞，并添哈克博士，博士Gouqing宋和吉尔科克女士威斯康星大学心血管生理学博士Peiman Hemmati（医学系，威斯康星大学麦迪逊分校）执行鼠标手术的核心设施。这项工作是由国家科学基金会通过一个研究生研究奖学金布莱恩弗里曼和美国国立卫生研究院R21 HL089679的支持。

1。捐赠者细胞转染<ol><li> 丰年间质干细胞（MSCs的，从H1胚胎干细胞Peiman Hematti博士惠赠捐赠所得;或者任何假设保险丝在体内的任何物种的细胞类型可受聘）在70 - 80％与1X胰蛋白酶合流（Mediatech 马纳萨斯VA 5分钟）。失活胰蛋白酶，α- MEM完全培养基（无抗生素，Invitrogen公司，卡尔斯巴德CA）18 。在300 XG离心5分钟。 </li><li>小心吸上清和重新暂停颗粒在1毫升的1X PBS和使用hemacytometer计数细胞。 </li><li>转让1.5 × 10 6细胞1.5 mL离心管中。在300 XG离心5分钟。 </li><li>小心吸上清液。悬浮颗粒在300μLR缓冲（霓虹灯转染系统，Invitrogen公司）和6微克（2微克/ 5.0 × 10 5个细胞）的P231 pCMVe betaAc一站式LUC（Addgene，剑桥，麻省）。将3毫升的E缓冲器（Invitrogen）电击到每个制造商的协议的对接端口（霓虹灯转染系统，Invitrogen公司）。 </li><li>转移细胞质粒的解决方案，以100μL霓虹灯吸管尖和electroporate与脉冲持续时间为20毫秒和1500伏级。广场电穿孔细胞成15 mL锥形管含有9.7 mL的α- MEM完全培养基。 </li><li>重复步骤1.5两个额外的时间和池转染细胞产生一个10毫升的总量。一个T175的α- MEM完全培养基含10 mL的容量瓶中加入10 mL细胞悬液（1.5 × 10 6细胞）。细胞的生存能力电击后约30％，产量约4.5 × 10 5％T175活菌。 </li><li>更改α- MEM完全培养基24小时后转。 </li><li>收获转染细胞时70 - 80％的融合（1〜2 - 3天之后，电击）。执行使用hemacytometer细胞计数，重悬细胞浓度为1.0 × 10 6cells/50μL的α- MEM完全培养基。花最少的时间细胞悬浮液中，在注射之前，以减少细胞死亡。 </li></ol> 2。内注射<ol><li>麻醉诱导异氟醚（凤凰制药公司，圣若瑟，密苏里州）工程组成的每一个细胞中的表达 Cre重组酶（B6.C - TG（CMV - CRE）1Cgn / J杰克逊实验室，巴尔港的转基因小鼠， ME）。 </li><li>从胸前地区取下的头发使用卷发器或化学头发卸妆。 </li><li>插管与18号导管（碧迪公司，新泽西州富兰克林湖）和地方对小鼠呼吸机在120 - 每分钟130次与搏出量为150μL。 </li><li>整个第四肋间空间，从而产生一个开胸外侧切口。 </li><li>可视化心脏，使转染细胞悬液25μL，用1 mL注射器（Temuro医药公司，萨默塞特，新泽西州）和28号针头（Bect的注射迪金森与合作，新泽西州富兰克林湖）。为了缓解内注射，防止过度损害的器官，弯曲的针头〜90度。 </li><li>注射后，使用可吸收缝合线（例如，薇乔）接近肋骨和肌肉层。缝合皮肤封闭，用4-0尼龙或丝​​绸。 </li><li>允许鼠标恢复从麻醉拔管。 </li><li> 对照组应包括的Cre小鼠接受中等注射只，CRE小鼠接受未转染细胞和转染细胞（另外，CRE小鼠接受不容易转染细胞融合）的野生型小鼠接受同样的浓度 。 </li></ol> 3。生物光子成像<ol><li>五到十五分钟前，腹腔成像（IP）的注入10μL每克的小鼠体重15毫克/毫升的D -荧光素（卡尺生命科学，霍普金顿，马）。 </li><li>诱导小鼠通过异氟醚麻醉诱导一个4％D 1 - 2％进行维修。 </li><li>广场小鼠仰卧在成像盒口罩管理1 - 2％异氟醚维护麻醉（精诺真生物光子成像系统，霍普金顿，马）。几个老鼠可同时成像。图像深水实验小鼠为便于比较，荧光信号的控制鼠标。 </li><li>使用生活图像软件（精诺真），设置适当的曝光时间（通常为60秒，看到的结果）。设置面积的图像，以适应老鼠，并保持整个成像区保持一致，以防止敏感性的变化。设置受到高度在4.5厘米。 </li><li>视野内取得相应的鼠标或鼠标的发光强度信号，并保存未修改图像文件。处理图像去除背景信号，相应的控制或unmanipulated鼠标。上述背景下的强度值对应内的动物细胞融合。生活图像软件（精诺真）或开源的图像分析软件，可进行强度分析。 Typically，一个感兴趣区域（ROI）是选择比较动物和实验之间的强度数据。 </li></ol> 4。代表性的成果要确定精诺真生物光子成像系统的灵敏度，组成表达荧光素酶（LUC - 231 - D3H1，精诺真）被送到C57/Bl6小鼠（杰克逊实验室）心肌细胞系的细胞注射浓度为 1 × 10 6，1 × 10 3，或1 × 10 1细胞细胞交付六个小时后，老鼠被注射用荧光素腹腔和使用的精诺真系统成像。 1000细胞（2 6小鼠成像， 图2）可以检测到一个特定的信号，但检测与万细胞（6小鼠成像6）更可靠。更重要的是，这项研究还有助于建立一个荧光素酶的表达和信号强度之间的粗糙的相关性。 和交付表达 Cre重组酶的小鼠心肌。大约一周后细胞交付，小鼠首次使用无精诺真系统的D -荧光素注射成像。正如预期的那样，没有酶底物，，没有强度信号检测（图 3 ）。接下来，D -荧光素腹腔注射和相应的信号强度，以荧光素酶，从而在两个，四个测试小鼠检测细胞融合。类似的信号检测到一个星期后（ 图3），提示MSC耦合 ​​融合产品，可在体内维持在这种情况下，这项研究被终止，以便评估心脏及周围组织，而是一个可以预见的较长期的分析和更频繁的成像跟踪维护，扩散ð也许迁移融合的产品，在小鼠体内。 <img alt="图1" src="/files/ftp_upload/3581/3581fig1.jpg" /> 图1。 检测体内细胞融合技术的原理，如果融合之间的Cre表达小鼠细胞和移植的细胞表达一个floxed荧光素酶质粒发生，荧光素酶将表示。可检测到荧光素酶注射到老鼠的D -荧光素酶的底物，然后成像鼠标使用精诺真生物光子成像系统（改编来自19个） <img alt="图2" src="/files/ftp_upload/3581/3581fig2.jpg" /> 图2。检测的灵敏度荧光素酶表达在心肌组织细胞与生物光子成像。细胞系组成表达荧光素酶（LUC - 231 - D3H1，精诺真）被送到C57/Bl6在不同的总细胞数的小鼠心肌内空间。重新小鼠的代表图像ceiving 1 × 10 6，1 × 10 3和1 × 10 1细胞（左到右）所示，成像进行了注射后约6小时。 <img alt="图3" src="/files/ftp_upload/3581/3581fig3.jpg" /> 图3。 在体内的发光细胞融合的指标量化 。MSCs的转染的LoxP位停止LoxP位荧光素酶质粒和交付表达Cre重组酶的小鼠心肌。大约一个星期，两个星期后，细胞传递，CRE小鼠成像利用精诺真生物光子成像系统，测量表明细胞融合的发光强度。 （一）覆盖的照片和1-4深水和小鼠的发光强度（左到右）17日天后细胞交付。 （二）1-4深水和小鼠的发光强度（左到右）细胞交付后的17天。一个选择感兴趣的区域（黄色）相应的注射部位和强度升使用ImageJ（免费源）软件20 evels。 （三）发光强度是归所有的实验条件下相同的地区深水鼠标的兴趣。在一个星期后，老鼠3和第4条呈阳性的发光信号，表明了小鼠细胞和移植的MSC的自发融合。坚持3鼠标在两个星期的信号。要确定特定器官本地化的信号对应鼠标3，胸腔暴露的主要器官切除和成像。 （四）覆盖的照片和鼠标3的发光强度。注意本地化在小肠的信号强度。 （五）3鼠标发光强度。 （六）覆盖照片和假鼠标的发光强度。 （G）的假鼠标的发光强度。

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没有利益冲突的声明。

这里的方法允许的话，第一次，在生物识别离散细胞融合和时空分析，包括小动物。后续的生物光子图像分析的方法相结合的Cre - LoxP位重组。跟踪不仅是细胞与细胞融合的方法是适合，而且病毒的细胞融合等可以跟踪病毒感染的证明。图像分析是快速，有可能图像的多个小动物的同时，融合检测融合在其相应的微环境，特定的细胞伙伴的频率和LoxP位停止LoxP位的萤光素酶质粒转染效?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>霓虹灯转染系统</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> MPK5000 </td><td></td></tr><tr><td>霓虹灯100μL，包</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> MPK10025 </td><td>包含R和发送缓冲区</td></tr><tr><td> A - MEM粉末</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> 12000-022 </td><td></td></tr><tr><td>胎牛血清（FBS） </td><td> Hyclone公司，UT斯达康洛根</td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C - TG（CMV - CRE）1Cgn / J </td><td>巴尔港杰克逊实验室，ME </td><td> 006054 </td><td></td></tr><tr><td>胰蛋白酶10X </td><td>费希尔科学，森林草坪，NJ </tD&gt; <td> MT - 25 - 054 - CL </td><td></td></tr><tr><td> L -谷氨酰胺</td><td>费希尔科学，森林草坪，NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D -荧光素</td><td>卡尺生命科学，霍普金顿，马</td><td> 122796 </td><td></td></tr><tr><td>精诺真生物光子成像系统</td><td>卡尺生命科学，霍普金顿，马</td><td> IVIS频谱</td><td></td></tr><tr><td>钠Biocarbonate </td><td>西格玛Aldrich公司，圣路易斯，密苏里州</td><td> S6014 - 500G </td><td></td></tr><tr><td>非必需氨基酸</td><td> Invitrogen公司，卡尔斯巴德，CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

已使用两个或两个以上的相同类型的细胞融合的能力，在整个进化的后生动物，形成了许多复杂的器官，包括骨骼肌肉，骨骼和胎盘。现代研究表明，同一类型的细胞融合赋予了增强的功能。例如，当胎盘保险丝的滋养层细胞形成的合体，合体能够更好地运输营养和激素跨比 1-4非稠合滋养的母胎屏障。最近的研究表明，不同类型的细胞融合可以直接细胞的命运。曾经被认为“回归”或修改融合细胞的命运是有限的细胞培养系统。但干细胞移植的到来我们发现，干细胞可以在体内与体细胞融合，融合促进干细胞分化 5-7 。因此，细胞融合是一个监管过程中Capable促进细胞的存活和分化，从而可以发展，修复组织和疾病的发病机制的极端重要性。 限制细胞融合的研究，是缺乏适当的技术：1）准确地识别融合的产品，以及2）随着时间的推移跟踪融合产品。这里我们提出一种新的方法来解决双方的局限性，通过融合后诱导生物发光（图1）;可生物发光检测灵敏度高，在 体内 8-15，我们利用一个构造编码萤火虫荧光素酶基因（Photinus pyralis）放在相邻一个终止密码子两侧LoxP位序列的细胞与细胞表达Cre重组酶蛋白基因的保险丝时，LoxP位网站裂解，使荧光素酶转录停止信号切除，由于信号是induciBLE，假阳性信号的发病率是很低的。与现有方法利用CRE / LoxP系统16，17不同的是，我们已成立了一个“活”的探测信号，从而承受的首次机会跟踪体内细胞融合的动力学。 为了证明这种方法，无处不在表达Cre重组酶的小鼠干细胞转染与构造来表达一个floxed终止密码子的荧光素酶的下游接受者。干细胞移植通过内注射和移植活体图像分析后进行跟踪存在随着时间的推移的心脏和周围组织的融合产品。这种方法可以适应在任何组织类型分析，在任何阶段的发展，疾病或成人组织修复细胞融合。

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

一个在生活随着时间的推移的生物体细胞融合的方法来跟踪描述。该方法利用CRE -<em&gt; LoxP位</em&gt;重组荧光素酶表达诱导细胞融合后。产生荧光信号可以检测到使用生物光子成像系统的检测〜1000细胞周围组织的敏感性，在生活的有机体。

阿的Cre - LOX P为检测细胞融合重组方法在体内</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K 次观看.  University of Wisconsin-Madison. 一个在生活随着时间的推移的生物体细胞融合的方法来跟踪描述。该方法利用CRE - LoxP位重组荧光素酶表达诱导细胞融合后。产生荧光信号可以检测到使用生物光子成像系统的检测〜1000细胞周围组织的敏感性，在生活的有机体。

视频: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

生物分子检测的干涉反射成像传感器（IRIS）

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

科研

生物工程

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. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

无标记技术的单细胞分泌物的时空成像

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

中空纤维生物反应器的<em&gt;在体内</em&gt;般的哺乳动物组织培养

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...