这个项目是由美国伊利诺伊州公共卫生部和美国国家科学基金会（DBI - 0852416）。

1。设备功能<ol><li>缺氧插入装置包含6个支柱，窝成一个标准的6孔板。气体流量为支柱，横跨在微流体网络的支柱基地，并流出设备的后面。在基地的底壁形成的微的支柱，是一个100微米厚的透气性PDMS膜，允许氧气扩散之间的气体微和文化传媒。因此，该器件通过建立所需的值，驱动器对媒体的氧气浓度的浓度梯度。 </li><li>该器件提供一个缺氧室和其他细胞充氧系统的优点：1）最大限度地减少了氧的扩散路径，允许更快的时间控制，2）提高超过充氧的空间控制，依赖的支柱微设计，3）拥有实验室占地面积较小，以提高实验效率和更高的吞吐量，以及4）适应常见的细胞培养工具（如多孔板），没有专门知识和设备的要求。 </li></ol> 2。设备制造<ol><li>第一支柱阵列与PDMS的，在以前的加工Delran模具成型的副本。 </li><li>下一步气体微在每个支柱的底部是采用标准的SU - 8的光刻和PDMS副本成型。 </li><li>透气膜制作在硅片上的PDMS的定义纺纱，以达到所需的厚度。在这个例子中，我们使用一个100微米厚的膜，这是由旋转500转10秒，30秒900转捏造。 </li><li>所有组件粘合在一起的设备（BD - 20型，电技术学校产品）与手持式等离子氧等离子体处理后。 </li></ol> 3。设备设置<ol><li>轻轻插入设备入板，一定要避免气泡。垂钓过程中插入的设备，有助于驱逐气泡的一面。对于实际的细胞为基础的实验，这一步必须做的无菌层流罩内。一旦设备被插入污染的机会大大减少，因此可以进行大会在非无菌环境中的孵化器。 </li><li>从源头上煤气罐连接管设备的进口和出口。基于细胞的实验中，文化的孵化器应该有一个洞，允许管的入口和油管应携带该设备后，放入孵化器的连接。要小心，以避免在设备上，可变形的PDMS足以粉碎底层细胞多余的压力。 </li><li>确保精密流量调节器关闭前开舱的最高监管机构，以避免溢出设备。启动气流量。轻轻打开精密流量调节到所需的流速（50-100毫升/分钟）。在接下来的一个小时密切注视值，并根据需要进行调整，因为压降会改变系统达到平衡，改变流速。为了避免在媒体中的气泡的形成，降低流速10-20毫升/分之间，具有较高的流量在经过最初的15分钟平衡时间。 </li><li>所需的实验持续时间后，停气流量，取出盘子，和相应的处理细胞（如裂解，染色，计数等）。 </li></ol> 4。设备验证<ol><li> 校准 <ol><li>选择的职位的数量和位置上的荧光氧 （FOXY）传感器幻灯片（根据审定要求），将使用氧气测量。幻灯片包含一个荧光钌染料涂层是由氧淬火。 </li><li>公开幻灯片直接为0，10，21％从煤气罐的氧气和适当的平衡5分钟后捕获的图​​像。 </li><li>出口依赖的荧光强度为每个位置和情节氧合浓度平均图像强度。 </li><li>生成校准曲线线性曲线拟合的0-10％和10-21％的线路。 </li></ol></li><li> 异质性 <ol><li>建立多点，横跨在定义的时间间隔（例如每1毫米）的通道宽度。请注意可能存在的间距取决于图像的重叠。 </li><li>通过设备的氧气浓度测量以及表面</li></ol></li><li> 平衡 <ol><li> FOXY幻灯片上选择三个点来衡量的氧气浓度。 </li><li>后的第一个图像捕捉周围的氧含量，立即打开精密流量调节器，开始进入设备的气体流量。在适当的时间间隔捕获的图像，以评估氧气concentr的时间长短和程度ATION平衡（如30分钟，每10秒）。 </li></ol></li></ol> 5。应用<ol><li> 伤口愈合 <ol><li>实验前的一天，浸泡在无血清培养基灭菌的PDMS的插入，以减少抑制天然气井的泡沫形成。 </li><li>培养细胞至100％，在6孔板汇合。 </li><li>建立与P200枪头来模拟伤口的单层直线划痕。 </li><li>吸出细胞介质，用5 mL媒体冲洗，并吸一次。重要的是不要扰乱细胞单层。 </li><li>笔芯的井4毫升的无血清培养基，降低细胞增殖。 </li><li>插入井，每口井，并连接到相应的氧气浓度。 </li><li>放置6孔板在37 ° C加热阶段的插入</li><li>捕捉时间的推移细胞图像所需的时间间隔和总工期。 MATLAB的T_Scratch，伤口测量算法，可用于分析不愈的表面积。 </li></ol></li></ol> 6。代表性的成果<ol><li> 设备验证 缺氧插入设备表现出了很大的改善，对氧平衡时间和程度的低氧室需要不到2分钟，以稳定在0.5％的氧气。设备膜井底差距的大小决定平衡效率的关键因素，具有较大的差距，需要更多的时间来达到稳定状态的氧浓度值。该器件还允许一个很大的控制空间单井充氧，形成多个条件允许，甚至产生以及表面上的一个循环的氧气配置文件。 </li><li> 伤口愈合 随着时间的推移单层细胞暴露于10％或21％的氧气和表面积的伤口进行了分析。 21％的氧气接触到的划痕封闭最慢和最快的在10％。图1显示了一个超过17小时的过程中划伤的图像。图5中的图表显示了开放的伤口面积％的氧气浓度在实验期间。 </li></ol> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1695/1695fig1.jpg"><img src="/files/ftp_upload/1695/1695fig1tmb.jpg" alt="图1" border="0" /></a> 图1说明设备功能的原理图和图。氧气插入设备制造由传统的光刻技术（微流控网络），副本成型（微流体网络和插入支架），并定义的PDMS纺纱（气体渗透膜）。一）嵌套到6孔板中的氧气设备。 B）24和96孔柱阵列的例子。 c）在一根柱子的截面示意图。氧流入的入口和设备，通过一个微网络上传播的支柱底部。整个透气PDMS膜，氧气可以自由地弥漫在支柱底部，并溶入培养基。 D）显微镜图像显示一个单通道的支柱，从上面的各种功能，保税平衡研究玻璃职位。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1695/1695fig2.jpg"><img src="/files/ftp_upload/1695/1695fig2tmb.jpg" alt="图2" border="0" /></a> 图2氧传感器设备的验证。以及在每个氧气张力的特点是使用一个平面的钌氧传感器。所有氧气混合物中的媒体缓冲平衡氮和5％的CO 2。 a）画出说明后高的效果，从而氧气的细胞膜和细胞之间的扩散距离，平衡时间和效益。高地建立了绑定到设备底部的玻璃切割职位。所有这三个职位大小产量平衡时间，大大改善了在低氧室。请注意，时间日志规模。二）情节描绘的0.2毫米的差距设备的快速氧平衡的响应时间。 c）多位置linescans还采取了根据微整个井，以确保同质化设备引进的氧气浓度。图描绘注入0％，10％和21％氧气10分钟后，测量氧浓度。四）设备有效地保持5天的10％以上的氧气。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1695/1695fig3.jpg"><img src="/files/ftp_upload/1695/1695fig3tmb.jpg" alt="图3" border="0" /></a> 图3更复杂的氧气微设计的实验。一）微双条件设置，产生一个稳定的0％和21％，超过14天的氧气的个人资料。 b）在500微米的宽度微叉和曲折的模式扩展到日在一个循环的氧气配置文件发送支柱结果。请注意该数据只描绘了一个有代表性的试验微对齐困难。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1695/1695fig5.jpg"><img src="/files/ftp_upload/1695/1695fig5tmb.jpg" alt="图4" border="0" /></a> 图4。Timelapse图像伤口缝合0，7，和17小时后最初从头。细胞交付整个实验持续时间21％的氧气。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/1695/1695fig6.jpg"><img src="/files/ftp_upload/1695/1695fig6tmb.jpg" alt="图5" border="0" /></a> 图5。伤口愈合率在从头检测氧气浓度的影响。

设备制造标准的SU - 8光刻，副本成型，并定义了纺纱和完全的聚二甲基硅氧烷。天然气是引进设备，建立了微支柱和文化传媒之间的浓度梯度，驾驶系统，对所需的平衡氧浓度。该设备已被证明能够有效调节的时间和空间内做好充氧，以及调节细胞行为适当。充氧的空间图案是由微定义在基地的支柱，因此，可以在制定光罩实施各种设计。此外，输液所需的气体进入井气相有望改善平衡时间和缺氧的程度。微流体混合网络可以适应的设备，提供了一种方法，只有少数股票煤气罐生产新型气体混合物。最后，媒体交流的机制将消除从多孔板，其中的细胞的反应可能需要删除该设备。 该设备已在体外或体外实验需要控制氧气浓度任何应用程序。由于氧气是一个重要的生理的变量影响的信号转导通路的绝大多数，是有限的研究领域，这将有利于创造力的研究员。这将有利于提高氧气浓度的时间控制的一些领域，包括癌细胞转移，睡眠呼吸暂停，并在许多其他的心肌缺血再灌注损伤。例如，间歇性低氧已与更多的浸润性癌，上调的metastastis相关基因的数量相对持续的低氧和常氧。空间的控制也很重要，因为氧气梯度发展，肝功能区划，药物的毒性，和干细胞小生境的关键。本文介绍的设备，将有利于通过提供实验室占地面积较小的，相对简单的操作要求，并大大增加细胞对氧接触到控制系统的研究领域。

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<td>PDMS-Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Planar FOXY sensor</td>
<td>&nbsp;</td>
<td>Ocean Optics</td>
<td>FOXY-SGS-M</td>
<td>Coated microscope slide</td>
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<tr>
<td>Gas regulator</td>
<td>&nbsp;</td>
<td>Omega</td>
<td>FL-1472-G</td>
<td>&nbsp;</td>
<tr>
<td>Gas</td>
<td>&nbsp;</td>
<td>Airgas</td>
<td>Custom mixes</td>
<td>All have 5% CO2</td>
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<td>SU-8 2150</td>
<td>&nbsp;</td>
<td>Microchem</td>
<td>&nbsp;</td>
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<td>MDCK Growth Medium w/ L-Glutamine</td>
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<td>30-2020</td>
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<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
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<td>T4049</td>
<td>&nbsp;</td>
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<td>L-Glutamine solution</td>
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fabrication and operation of an oxygen insert for adherent cellular cultures 

氧气是许多细胞途径的关键调制器，但目前的设备允许<em&gt;在体外</em&gt;氧气调制失败，以满足生物医学研究的需要。低氧室提供了一个简单的系统，以控制在标准的文化船只充氧，但缺乏精确的时间和空间，在细胞表面的氧气浓度控制，防止各种生理现象的研究及其应用。其他系统改善缺氧室，但其运作需要专门的知识和设备，使他们平均研究员恐吓。一个微型插入多孔板已经开发更有效地控制时间和空间的氧气浓度，以更好的模型发现的生理现象<em&gt;在体内</em&gt;。该平台由一个聚硅氧烷插入巢成一个标准的多孔板和被动的旨在气体渗透膜的微流控燃气管网供应，调节氧气输送到贴壁细胞。该设备使用简单，连接到提供的压力引入到平台所需的氧气浓度的气瓶。制造涉及到一个标准的SU - 8光刻，副本成型，并在硅片上的旋转定义的PDMS组合。保税后的表面，使用手持式等离子系统治疗设备的组成部分。验证是通过平面荧光灯的氧传感器。平衡时间是为了分钟的氧气型材多种，可以实现基于设备的设计，如在这项研究中所取得的循环轮廓，甚至氧梯度模仿这些发现<em&gt;在体内</em&gt;。该设备可消毒细胞培养，使用常用的方法，而不丧失功能。该设备的适用性研究<em&gt;在体外</em将展示&gt;伤口愈合反应。

Watch this Scientific Journal Video about Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures at JoVE.com

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

制造和验证一个附加的平台，在空间和时间在6孔板充氧提供增强的控制。该设备是适应了文化系统，可用于调查的氧气对伤口愈合的影响。

为贴壁细胞培养的氧气插入的制备及操作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

fabrication-operation-an-oxygen-insert-for-adherent-cellular

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

9.7K 次观看.  University of Illinois. 制造和验证一个附加的平台，在空间和时间在6孔板充氧提供增强的控制。该设备是适应了文化系统，可用于调查的氧气对伤口愈合的影响。

视频: Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大型插入环境基因组文库生产

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

科研

生物学

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量:贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

检测，可视化和定量活性氧的生成在变形虫模型系统

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

在细胞间接触缺失的两个蜂窝型的插入共培养系统的开发

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...