The authors thank Mike Myles, Sam Saunders, and C.W. Elton at the ECU Multimedia &amp; Technology Services for providing assistance in video production. We acknowledge the grant support from North Carolina Biotechnology Center, Golfers against Cancer, Brody Brothers Endowment Fund, American Heart Association, and ECU/Vidant Cancer Research and Education Fund (L.V.Y. and M.J.R.).

1，细胞培养伤口闭合含量<ol><li>使用0.25％胰蛋白酶-EDTA溶液中的组织培养板中分离细胞。沉淀的细胞在15毫升锥形管中，离心，吸出上清液，并重新悬浮的细胞在培养基中。铠细胞在6孔板的适当数量为在24小时内100％汇合。 注意：测试可能需要确定时间和细胞数达到100％汇合时，由于细胞的类型和该井所使用的大小（例如，1×10 6 B16F10黑素瘤细胞接种在一个良好的6孔板前24小时至伤口代）。可替换地，可以使用12孔或24孔板中。 注：如果可用，细胞可在处理过的细胞培养板使得伤口预铸被接种到文化 - 插入（从ibidi有限责任公司）。文化 - 插入可以通过枪头保护组织培养板的表面不受损坏。如果一个企业文化 - 插入时忽略步骤1.2。 </li><li>在无菌的环境（通常是生物安全罩）用200μl的移液管尖，以牢固地压在组织培养板的顶部，并通过细胞单层迅速地使一个垂直向下的伤口。不同尺寸的移液管尖端可以用来使所期望的伤口大小。不要过度使用武力的组织培养板吸头，因为它会损坏表面。如果伤口预铸，该步骤可以省略。 </li><li>小心吸媒体和细胞碎片。慢慢地对孔壁，以覆盖所述孔的底部，并避免额外的分离的细胞补充足够的培养基。以下伤口的产生和检测的初始图象，应采取。将组织培养板在培养箱中设定在适当的温度和CO 2浓度（通常为37℃和5％CO 2）。 </li><li>在几个时间点， 例如每3小时从incubat除去板或者，并将其放置在倒置显微镜下拍摄快照的图片，并检查伤口闭合。如果时间推移显微镜可用，拍照分钟的每隔X数量（通常以每5分钟为单位）持续时间在温度和CO 2控制腔。取决于细胞类型，伤口闭合的时间可能会发生变化。 </li><li>要分析的快照图片的结果，测量在伤口的一侧到另一使用比例尺的距离。用散点图或条形图， 例如 图1分析，并明确目前伤口闭合随着时间的推移。 </li><li>为了分析时间推移的图片，导入图片到ImageJ的软件（可自由查看的http://rsb.info.nih.gov/ij/）进行视频。要做到这一点打开ImageJ的，点击文件，导入，和图像序列。找到图片文件;单击该文件中的第一张照片。保存视频选择文件，另存为，和AVI。单个细胞的迁移可观察到恰拉cterize定向运动。此外，形态特征，如伪足形成和后缘回缩可以研究以及（ 图2和辅助视频）。 </li></ol> 2，Transwell小细胞迁移和入侵检测<ol><li>在无菌的环境（通常是生物安全罩）使用离心非酶细胞解离缓冲液，或0.25％胰蛋白酶-EDTA溶液中，沉淀的细胞分离的组织培养板中的细胞，并吸出的现有媒体留下沉淀的细胞。重悬的细胞在含有0.1％BSA（牛血清白蛋白）的无血清的细胞培养基。 注意：根据该细胞系被调查的0.25％胰蛋白酶-EDTA会影响细胞迁移和侵袭由于各种受体在细胞表面17的切割。 注意：每毫升的细胞数依赖于细胞的大小。进一步的测试，可能需要发现亲的接种密度志愿组织的最佳效果。通常为1×10 6个细胞/毫升是一个很好的起点，大多数细胞类型，当使用24孔Transwell插入。有一系列的transwell插入物的大小并增加细胞的数量应调整为相应。 </li><li>板100μl的细胞溶液在一个Transwell小插入过滤膜的顶部，并孵育10分钟，在37℃和5％CO 2，以使细胞沉淀下来。 注意：使用的transwell小室的膜的特定细孔径是依赖于细胞的样品中的各个尺寸。例如，如果该细胞是太小它们可以穿过的孔，而不需要促进迁移，或者如果它们太大，他们可能没有通过毛孔适合。有几个的transwell插入物与孔径大小的范围从3微米，5微米，8微米的用于细胞迁移测定。 Transwell小刀片是可商购自例如康宁公司。 注：Transwell小米igration分析可以很容易地修改，以执行细胞侵袭实验。要做到这一点，添加细胞外基质（ECM）材料上的Transwell小膜的顶部，然后对ECM的顶部添加细胞。例如，基质胶解冻和液化在冰上，然后30-50微升基质胶加入到24孔Transwell小插入和固化在37℃的培养箱中培养15-30分钟，以形成一个薄的凝胶层。细胞溶液在基质胶涂层的顶部加入，通过细胞外基质，以模拟的入侵。 注意：有Transwell小细胞迁移和Transwell小细胞侵袭实验之间有明显的差异。 Transwell小细胞迁移实验测定细胞走向化疗引诱的趋化能力。 Transwell小细胞侵袭测定法，但是，通过细胞外基质的同时测量细胞趋化性和侵袭的细胞，即通常在癌症转移或胚胎发育的发现过程。 </li><li>使用移液管，非常caref来自ully添加600微升所希望的化疗引诱到下部腔室在一个24孔板的底部。添加化学引诱而无需移动Transwell小插件，避免产生气泡。确保在底部的井的化疗引诱液，使接触带的膜在上部以及形成趋化梯度。温育时间是依赖于细胞类型和化学引诱被使用。 注：进一步的测试可能需要确定潜伏期。 注意：对于贴壁细胞，迁移的细胞将附着在膜1,8的另一侧。迁移的细胞进行定量可以执行以下步骤2.4到2.8（步骤2.4-2.8不需要在无菌环境中进行的）来执行。对于非粘附细胞，迁移的细胞将下降到介质中的下腔室。迁移细胞的数目可以通过使用血细胞计数器或算流式细胞仪5。 </li><li>从板块中删除Transwell小插件。使用棉签多次需要小心地取出，但没有从膜的顶部而不损坏它迁移媒体和剩余的细胞。 </li><li>加600-1,000微升70％乙醇到24孔板的孔中。将transwell小插件放入70％乙醇中10分钟，以使细胞固定。从24孔板取出Transwell小插入和使用棉签从膜的顶部取出剩余的乙醇。允许的transwell小膜干燥（通常10-15分钟）。 </li><li>加600-1,000微升0.2％的结晶紫为24孔板的孔和膜定位成它用于染色。在室温下孵育5-10分钟。 </li><li>轻轻地从膜的顶部用移液管尖端或棉棒取出结晶紫。非常小心，以避免洗去固定的细胞，可以根据需要沾膜进入蒸馏水多次以除去过量的晶体VIOLE万吨。允许的transwell小膜干燥。 </li><li>视图下方倒置显微镜和计数细胞在不同视场的数目，以得到通过膜迁移朝向化学引诱并附着在膜的下侧（ 图3）细胞的平均总和。 </li></ol>

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The authors declare that they have no competing financial interests.

细胞迁移是在癌症研究，研究的一个重要方面，它也可以应用到发育，免疫和伤口愈合研究。细胞培养伤口闭合试验和Transwell小细胞迁移和侵袭测定法揭示了细胞迁移性的行为的详细信息，并且可以用于研究细胞迁移1,2,10,14的分子机制。我们的研究中使用这些细胞运动性测定法来确定B16F10黑色素瘤细胞系的迁移速度和侵袭能力。 细胞伤口愈合试验检查一个特定细胞系...

蠕动是活细胞的一个重要特征。细胞迁移是参与生命，胚胎发育，免疫反应，以及许多病理过程，如癌症转移和炎症1-9的概念。因此，方法来研究细胞的迁徙行为是非常有用的研究工具，在生物医学科学，生物学，生物工程及相关领域学科门类齐全的。 在癌症研究中的细胞迁移的研究是特别感兴趣，因为死亡的癌症患者的主要原因是关系到转移进程。为了让癌细胞扩散和传播遍及全身，癌细胞必须迁移和入侵，通过细胞外基质（ECM），intravasate进入血液循环，连接到一个远程站点，最后渗出，形成远处病灶1,10〜12。各种生物的方法可以用来详细研究这些事件。细胞培养伤口闭合的D中的Transwell小迁移和入侵检测被广泛应用于科学界1,10。这些测试可以提供必要的数据，可能会允许对如何以及特定细胞类型可以自发地迁移或响应一个化疗诱和定向朝向其迁移的理解。几个迁徙的表型进行了说明。细胞可以迁移见于间质或变形虫样运动或标记的集体迁移或细胞流13多细胞运动的单细胞形式如。在游动细胞用于运动的方法，可以使用细胞培养物的伤口闭合测定法可以容易地观察到。 在众多的方法来研究细胞迁移，细胞伤口缝合法是最简单的一种。这种方法可用于确定整个细胞团的迁移能力。时采取的一个步骤进一步它可以用于迁移14时，观察各个细胞的形态特征。随着伤口闭合的分析很多表型可能显露出来。比较控件时测量近距离随着时间的推移可能会发现特定迁移量的变化或受损迁徙的表型是前所未知的。此外，单细胞lamellipodium形成，尾部回缩，并定向运动可能会给什么可能会削弱或增强的利息14细胞的线索。 Transwell小迁移和侵袭测定法可用于分析单个细胞的定向到不同的化疗诱响应它们是否是趋化因子，生长因子，脂质，或核苷酸4,5,8,15,16的能力。它也可以评估差分迁移能力因过度表达的受体1,14的。这些测定法还可以用于鉴定和表征细胞迁移的关键调节剂，如Rho家族的小GTP酶2。下面的这些短且容易存取权限sible测试中，细胞迁移的模式和小区的侵入成三维基质的能力也可以被确定。

<table border="0" cellpadding="0" cellspacing="0" width="601">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">11995-073</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Fetal bovine serum (FBS)</td>
			<td style="width:133px;">Gemini</td>
			<td style="width:107px;">100106</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Bovine serum albumin (BSA)</td>
			<td style="width:133px;">Sigma-Aldrich</td>
			<td style="width:107px;">A4503-50G</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Trypsin EDTA 0.25%</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">25200-056</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Dulbecco&#8217;s phosphate buffered saline (DPBS)</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">14190-250</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:169px;">Crystal violet</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:107px;">C0775-100G</td>
			<td style="width:193px;">Dissolved in water at 0.2%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Cell disassociation buffer</td>
			<td style="width:133px;">Gibco</td>
			<td style="width:107px;">13151-014</td>
			<td style="width:193px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">Cell culture incubator</td>
			<td style="width:133px;">Thermo Fisher Scientific</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # 3145</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">SterilGARD biosafety hood</td>
			<td style="width:133px;">The Baker Company, Inc.&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # VBM-600</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:169px;">EVOS Fl inverted microscope</td>
			<td style="width:133px;">Thermo Fisher Scientific</td>
			<td style="width:107px;"></td>
			<td style="width:193px;">Model # AMF-4302-US</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:169px;">Tissue culture plate</td>
			<td style="width:133px;">Becton Dickinson</td>
			<td style="width:107px;">353046</td>
			<td style="width:193px;">Catalog number varies depending on the type of culture plate</td>
		</tr>
		<tr height="42">
			<td height="63" rowspan="2" style="height:63px;width:169px;">Corning Transwell insert</td>
			<td rowspan="2" style="width:133px;">Fisher</td>
			<td rowspan="2" style="width:107px;">07-200-150</td>
			<td rowspan="2" style="width:193px;">Catalog number varies depending on the pore size of the membrane</td>
		</tr>
	</tbody>
</table>

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.

这里提出的伤口闭合试验和Transwell小细胞迁移测定法使用小鼠B16F10黑素瘤细胞作为模型系统进行。在伤口愈合测定法，B16F10细胞接种在6孔组织培养板中并生长至24小时内100％汇合。使用使用时间推移显微镜记录枪头和伤口闭合（细胞迁移）生成约700μm宽的伤。可替换地，伤口闭合，也可以通过采取快照图片在不同时间点，如果时间推移显微镜不可用1研究。四张照片的时间推移系列在0，4,8...

Watch this Scientific Journal Video about In vitro Cell Migration and Invasion Assays at JoVE.com

In vitro Cell Migration and Invasion Assays

常用的，非常方便的方法研究细胞迁移和侵袭的体外描述。第一种方法是测量细胞运动的细胞伤口愈合测定法。第二种方法是，评估的趋化和细胞的侵袭能力Transwell小迁移和侵袭实验。

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 ...

in-vitro-cell-migration-and-invasion-assays

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biology

In vitro Cell Migration and Invasion Assays

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

136.8K 次观看.  East Carolina University. 常用的，非常方便的方法研究细胞迁移和侵袭的体外描述。第一种方法是测量细胞运动的细胞伤口愈合测定法。第二种方法是，评估的趋化和细胞的侵袭能力Transwell小迁移和侵袭实验。 

视频: In vitro Cell Migration and Invasion Assays

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

化验粘附和酿酒酵母琼脂入侵

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

科研

生物学

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

利用磁调制生物传感系统的生物检测的快速均质检测

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

雪旺的星形胶质细胞使用相互作用分析<em在体外检测

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

体外聚集测定使用tau蛋白的蛋白质

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

为评估细胞Mechanosensing和Durotaxis简化系统<em&gt;体外</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

A Customizable Chamber for Measuring Cell Migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

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 ...