作者承认克莱姆森大学创意咨询程序和NSF CBET1254609为这个项目提供资金。

1，一种生产微通道室以产生浓度梯度<ol><li> 切割丙烯酸防霉片 <ol><li>获得一块所需宽度的丙烯酸。通常选用1/16英寸厚的压克力板材。较厚的片是难以切割好，非常薄的片材不具有必要的机械强度，使它们的过程中断裂或变形。 </li><li>创建与丙烯酸片的所需形状的CAD文件到聚二甲基硅氧烷（PDMS）内产生的空腔。调节通道的宽度和长度，以实现相关的个别实验方案所需的梯度。 <ol><li>在这里，用丙烯酸片尺寸如下:由0.127厘米宽的通道连接（0.254厘米长）两个正方形（0.254厘米点¯x0.254厘米）（ 图1）。 </li></ol></li><li>根据马努在激光切割机导入CAD文件到激光切割设备，然后将一块亚克力厂家的协议。 注:另外，3D打印从CAD设计作品。这种方法的优点是，替代材料，可用于在模具中;但是，分辨率和重复性，可以用3D打印相比，激光切割下降。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/55264/55264fig1.jpg" /> 图1:微通道商会计算机辅助设计（CAD）表示。该图像描述了创建微室所需的亚克力插入的CAD图纸。 1 A）CAD出图，1B）CAD的侧视图英寸的尺寸图，1C）CAD的顶视图英寸的尺寸图的顶视图<a href="http://ecsource.jove.com/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <ol start="2"><l我&gt; 浇注聚二甲基硅氧烷（PDMS） <ol><li>在一个称量船（重量）商业弹性体基的（ 如的Sylgard 184）与1重量份的商业橡胶固化剂（ 如的Sylgard 194）的混合10份。通常情况下，混合10克橡胶基地到1g弹性体固化剂。 </li><li>搅用微量5分钟的基料和固化剂相结合。 </li><li>设立一个真空钟，并将与PDMS中称量船在真空30分钟以除去截留的空气气泡。 </li><li>关闭真空并取出称重舟。倒入一个小培养皿丙烯酸切出顶部的PDMS。确保PDMS完全覆盖刀片。 </li><li>允许PDMS固化过夜（至少18小时）在室温下。 </li></ol></li><li> 去除PDMS插入 <ol><li>固化的PDMS后，通过小心地切割PDMS周围的丙烯酸系片用手术刀取下刀片。 </li></ol&gt; </li></ol> 2.电镀细胞在微通道商会<ol><li> 灭菌电镀细胞室中。 注意:虽然PDMS可使用环氧乙烷或其他技术进行灭菌，快速UV处理是快速，廉价，并且足以用于细胞培养研究。短UV处理时间也没有损害的PDMS片的结构或机械完整性。 <ol><li>在标准细胞培养柜，完全覆盖，用70％的乙醇的腔室。 </li><li>把与所述盖子关闭层流罩的培养皿。 </li><li>关闭引擎盖打开紫外线灯。暴露于UV光1-2小时。 </li><li>打开层流罩，并等待15分钟重新建立流动。 </li><li>除去70％的乙醇。 </li><li>用无菌磷酸盐缓冲盐水（PBS）洗涤腔室的两倍。使用1毫升的PBS每次洗涤。除去PBS并允许室与盖子关干通宵。 注:此外，相反使用层流罩中，使用的UV腔室箱灭菌如果有一个可用。通过将UV光源在一个纸箱用铝箔衬里的背面使紫外线框。铝箔反射光，以允许甚至照明样品。 </li></ol></li><li> 在室内电镀细胞 <ol><li>使用计数台盼蓝和血球细胞。 <ol><li>加入100μL细胞悬液400微升0.4％台盼蓝，轻轻混匀。由玻璃盖玻片下轻轻填充两院应用100微升此解决方案血细胞计数仪。 </li><li>使用显微镜用10倍的目标集中在血球电网。用一只手式计数器，以一组的血球16平方内计数活的未染色细胞。 </li><li>在全体4套16平方的细胞。从4台16平方取平均细胞计数和由5×10 4繁殖。这个最后的数字是numbe在细胞悬浮液中细胞/ mL的河</li></ol></li><li>添加2500细胞一个孔在每个室中。这转化为的〜30,000 / cm 2的近汇合的细胞密度。 </li><li>允许细胞在腔室坐60分钟添加介质之前（Dulbecco氏改良的Eagle氏培养基，10％胎牛血清，1％青霉素 - 链霉素）。 </li></ol></li></ol> 3.浸泡葡聚糖琼脂糖块<ol><li> 创建琼脂糖块 <ol><li>琼脂糖倒3％到3D印刷模具（2毫米×2毫米×2mm）的。 </li><li>允许琼脂糖在真空室内固化30分钟，以除去任何气泡。 </li><li>除去从模具琼脂糖块。 注意:可替换地，琼脂糖倒3％成小培养皿在厚度为2毫米。切使用手术刀或者其它切割工具2毫米×2mm的×2mm的块。这不是作为模具系统那样精确，但是它可以更容易一点做。 </li></ol></li><li>完全submergE在趋化因子的所需浓度的溶液琼脂糖块。为了评估浓度梯度的形成，荧光右旋糖酐第一冲洗室。葡聚糖的浓度和分子量应匹配的可溶性因子或感兴趣的药物。 </li><li>浸泡琼脂糖块过夜。 </li></ol>葡聚糖扩散4.时间推移，评估可溶性因子浓度梯度<ol><li>将葡聚糖浸泡琼脂糖块在微室的小方井。 </li><li>放置所述微通道室中的细胞成像系统，或使用其他荧光显微镜与时间推移的能力。根据制造商的说明开始的时间推移。 注:显示的结果采取GFP过滤器，50％的光，pH值4/10和放大4倍。 </li></ol>细胞生长5.定时<ol><li>板中的细胞微通道室中的一端室。在这里，使用3T3成纤维细胞。添加2500细胞一个孔在所述微通道室中。在2.2.1中描述计数细胞。 <ol><li>允许细胞在腔室坐60分钟添加介质之前（Dulbecco氏改良的Eagle氏培养基，10％胎牛血清，1％青霉素 - 链霉素）。保持与铺板的细胞在微室中于37℃在5％CO 2。通过信道细胞迁移可以用显微镜观察。 </li><li>添加标记或使用在腔室作为标记的显微缺陷，以作为一个起点，以量化的距离的小区前移动。 </li></ol></li><li>通过将琼脂糖凝胶（8 mm 3的块）包含建立梯度浓缩生长因子，趋化引诱剂，药物，或者被测试其他因子（这里，40％的FBS被用作一个例子）在所述微通道的一端室。 </li><li>就拿移动单元前的时间推移图像。在此，结果表明，该细胞的图像来回核苷酸已采取使用成像系统每12小时。 </li><li>使用电池前的照片来量化细胞的生长速度。计算由第一利用MATLAB polyfit功能（roipoly）来创建由细胞前，通道壁，和参考标记限定的多边形掩模的增长率。该掩模的尺寸，得到由此计算平均电池锋速度前的迁移距离。其他研究已经利用类似的方法8。 注:在该相同的距离进行比较的细胞生长前面各帧的可溶因子的浓度上的边缘。可溶性因子的浓度梯度是使用一维扩散模型近似计算。 </li><li>采取用鬼笔环肽和DAPI染色的细胞的荧光图像。 <ol><li>与鬼笔环肽染色之前修复细胞。 <ol><li>在37℃的温水4％多聚甲醛。 </li><li>补充足够的4％多聚甲醛覆盖细胞。保持在细胞中多聚甲醛10分钟。 </li><li>用PBS冲洗两次，每次15分钟。使用足够的PBS覆盖细胞。 </li></ol></li><li>从细胞中除去PBS中并添加足够的透化溶液（0.1％的Triton X-100的PBS），以覆盖细胞。离开溶液10分钟。 </li><li>用PBS洗两次，每次5分钟。使用足够的PBS覆盖细胞。 </li><li>从细胞中除去PBS中并添加20分钟阻断溶液（在PBS中1％牛血清白蛋白）。 </li><li>除去阻断溶液并用PBS洗涤2次，每次5分钟。使用足够的PBS覆盖细胞。 </li><li>从这点来说，在不使用时保护细胞免受光用铝箔。从细胞中除去PBS中并在PBS中添加鬼笔环肽488 1:50稀释1小时。 </li><li>洗2次在PBS中5分钟，每次洗涤。使用足够的PBS覆盖细胞。然后从细胞中移除PBS。 </li><li>在PBS 1000细胞15分钟:加入DAPI（4&#39;，6-二脒基-2-苯基）稀释1。 </li><li>删除和DAPI洗涤细胞两次，用PBS进行5分钟，每次洗涤。 </li><li>删除最后一次洗涤和补充足够的PBS覆盖细胞。细胞现在可以用荧光显微镜成像。 </li></ol></li></ol>

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(69), (2012).</li></ol>

作者什么都没有透露。

我们的微腔可用于目的，包括确定细胞迁移速率响应于生长因子和趋化因子以及从聚合物基质测定药物的扩散速率的多种。它可以利用我们的微腔室生长细胞并放置一个趋化在腔室的一端。细胞生长响应于趋化和细胞迁移可以通过取可在为了确定细胞迁移率分析的时间推移的图像进行量化。这种方法的代表性结果示于图3，其中被确定的增长率是使用MATLAB polyfit函数13.4微米/小时。 <...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

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	<tbody>
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			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
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		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
			<td></td>
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		<tr>
			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
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		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
		</tr>
	</tbody>
</table>

a customizable chamber for measuring cell migration

细胞迁移是免疫反应，生长和伤口愈合的重要组成部分。细胞迁移是一个复杂的过程，涉及到细胞，细胞外基质，和可溶的和不可溶的化学因素（ 例如 ，化学引诱物）之间的相互作用。标准方法测量细胞，如Boyden小室测定法中，通过在除法器的任一侧细胞计数工作的迁移。这些技术很容易使用;然而，他们提供针对不同应用的小几何修改。与此相反，微流体装置可用于观察与可溶性因子1,2定制浓度梯度细胞迁移。然而，用于制造基于微流体分析的方法是很困难的学习。 这里，我们描述用于创建细胞培养室，测量响应于化学浓度梯度细胞迁移的简单方法。我们的细胞英里格雷申室方法可以为了研究细胞迁移对于各种应用创建不同的线性浓度梯度。该方法是相对容易使用，并且通常由大学生进行。 所述微通道室通过将在最后的微腔的形状的丙烯酸插入井到培养皿创建。在此之后，聚（二甲基硅氧烷）（PDMS）倒在刀片的顶部。将PDMS被允许硬化，然后除去插入件。这允许以任何所需的形状或尺寸的创作孔中。细胞可以随后添加到微室中，和可溶性剂可以通过在所需的剂浸泡琼脂糖块被加入到孔中的一个。琼脂糖块加入到孔中的一个，和时间推移的图片可以量化细胞迁移可采取微通道室。变化，以这种方法可以用于给定的应用进行，使得这种方法HIG溶血素可定制的。

图2示出横跨通道的细胞前的响应于放置在信道的从细胞接种，其中相对端胎牛血清的梯度的运动。小区前示出在48小时（ 图2A），72小时（ 图2B），96小时（ 图2C），和120小时（ 图2D）交电镀。小区前的运动用这些时间推移图象跟踪，和迁移距离和生长率分别由MATLAB polyfit函数计算。由此看来，平均气泡锋速?...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration at JoVE.com

A Customizable Chamber for Measuring Cell Migration

该协议细节来测量响应于也可用于确定药物的扩散速度出聚合物基质的化学引诱物细胞迁移可定制方法。

定制室测定细胞迁移

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

a-customizable-chamber-for-measuring-cell-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.5K 次观看.  Clemson University. 该协议细节来测量响应于也可用于确定药物的扩散速度出聚合物基质的化学引诱物细胞迁移可定制方法。 

视频: A Customizable Chamber for Measuring Cell Migration

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

脑片的刺激，利用微流控网络和标准灌注腔

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

测量细菌细胞的抗弯刚度，使用光学陷阱

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.