Name: quantitative assessment of human neutrophil migration across a cultured bladder epithelium
Uploaded: 2013-11-07T19:40:00
Description: Watch this Scientific Journal Video about Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium at JoVE.com

这项工作是由卫生研究院（NIH）国家机构支持授予R01-DK080752和P50-DK064540。我们感谢J. Loughman为她建立这个实验的努力。

1。培养5637膀胱上皮细胞执行中制备，用紫外照射和擦拭用70％乙醇中的层流的组织培养罩下面的步骤。 <ol><li>制备含有10％胎牛血清（FBS）的RPMI称为+]的RPMI-1640培养基中，过滤消毒用0.22微米孔径的过滤器，并温热至37℃的水浴中。 </li><li>解冻5637细胞的离心管（美国典型培养物保藏HTB-9，膀胱癌派生）在37℃水浴。将解冻的细胞迅速转移到含20ml RPMI + 75 cm 2的组织培养...

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用培养的膀胱上皮细胞系和新鲜分离的人中性粒细胞中，我们建立了transuroepithelial中性粒细胞迁移的体外模型。这种模式一直在开始解剖过程中尿路感染（UTI）的先天免疫反应，一个极其常见的细菌感染通常是由统一企业9的复杂性。在膀胱或膀胱炎的感染，中性粒细胞招募到膀胱内腔是细菌清除10是必不可少的。建立在炎症反应在脸上的立足点，统一企业延缓PMN到膀胱，从...

细胞的整个身体的运动，往往跨越很长的距离，需要的生长发育，伤口愈合，以及免疫反应。细胞迁移是复杂的，需要许多不同的过程，包括信号通路和细胞骨架组分的重排的协调。细胞可以随机移动（化学激活），以及对特定的化学梯度（趋）。许多技术已被开发，研究细胞迁移的体外 。最古老和最常用的技术，在Boyden小室，由一个垂直的双室系统，其中趋化物质被放置在底室和感兴趣的细胞被放置在顶室1。细胞在整个可渗透过滤器的移动，以确定大小的孔，分隔两个室进行监测。附加的技术已被开发来研究细胞的迁移，包括Zigmond室2和唐恩室3。这些集体的方法已经取得了显著洞察到许多不同类型的细胞的移动。 除了询问化学激活和趋化的基本原理，双室测定法提供了便利细胞迁移通过细胞外基质成分和内皮细胞和上皮细胞层的调查。两腔系统相对于其它技术的优点在于，所述多孔膜可以涂覆有蛋白如胶原蛋白或纤维蛋白原，以及跨细胞外基质状屏障细胞的迁移可以被评估。此外，培养的细胞系可以生长和分化的可渗透支撑。研究细胞的跨内皮屏障的运动，培养的内皮细胞接种并生长在可渗透支撑件的上部贮存。运动细胞，如免疫细胞，加入到上水库和迁移到整个连接下水库在生理心尖至基底外侧方向内皮屏障被观察到。这种模式是非常宝贵的理解免疫细胞的外渗出来的血流。而相比之下，跨内皮迁移，细胞跨越上皮屏障的运动，通常会发生在基底外侧至顶端的方向。为了在体外模拟这些事件中，研究人员和种子上的可渗透支撑件的下侧生长培养的上皮细胞。游动细胞被添加到上面的储存和迁移穿过上皮屏障，较基底外侧至顶端的方向，被监测。跨膜迁移的这种模型已经显著有助于我们在粘膜表面的炎症反应，尤其是肺部和肠道4,5理解。 与此相反，通过尿路上皮免疫细胞的运输已经很少受到注意。让我们进一步understandi的纳克感染肾盂肾炎大肠杆菌 （UPEC）期间在尿路先天免疫反应，我们开发了一种在体外试验中，transuroepithelial中性粒细胞迁移测定中，使多形核白细胞的调查（PMN嗜中性粒细胞）的移动跨越膀胱上皮屏障6 -8。与跨膜迁移其他两腔模型，体外培养的人膀胱上皮细胞是生长在可渗透支撑底部，并形成合流上皮层。人中性粒细胞，从静脉血中分离，被施加到上皮细胞层的基底一侧，然后在生理学相关的基底外侧至顶端的方向穿过上皮迁移定量响应于感染大肠杆菌的不同菌株大肠杆菌或趋化分子的存在。许多研究侧重于中性粒细胞的运动，无论是化学激活和趋化作用，在没有额外的细胞类型。该transuroepithelial中性粒细胞迁移测定法是有利的，因为它考虑到感染期间膀胱上皮细胞和免疫细胞之间复杂的相互作用。这个听话的体外模型，以允许在尿路上皮表面免疫反应的详细调查的潜力。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Transwell Inserts (i.e.&#160;permeable supports)</td>
			<td>Corning</td>
			<td>3472</td>
			<td>6.5 mm, 3 &#956;m pore, polyester membrane insert</td>
		</tr>
		<tr>
			<td>BD Vacutainer Blood Collection Tubes</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>366480</td>
			<td>16 mm x 100 mm x 10 ml green cap tubes containing sodium heparin</td>
		</tr>
		<tr>
			<td>BD Safety-Lok Blood Collection and Infusion Set</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>367281</td>
			<td>21 G x 0.75 in needle x 12 in tubing</td>
		</tr>
		<tr>
			<td>BD Vacutainer one-use, nonstackable holder</td>
			<td>Becton, Dickinson and Company (BD)</td>
			<td>364815</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>D4876</td>
			<td>From Leuconostoc mesenteroides</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS density centrifugation solution</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment Plates</td>
			<td>Corning</td>
			<td>3473</td>
			<td>24-well, clear flat bottom</td>
		</tr>
		<tr>
			<td>N-Formyl-Met-Leu-Phe (fMLF)</td>
			<td>Sigma-Aldrich</td>
			<td>F3506</td>
			<td>Reconstituted in DMSO to 10 mM</td>
		</tr>
		<tr>
			<td>Recombinant Human CXCL8/IL-8</td>
			<td>R&#38;D Systems</td>
			<td>208-IL</td>
			<td>Reconstituted in PBS to 100 &#956;g/ml</td>
		</tr>
	</tbody>
</table>

quantitative assessment of human neutrophil migration across a cultured bladder epithelium

从外围到炎症部位的免疫细胞的募集是在任何粘膜表面的先天免疫反应的重要步骤。在膀胱感染，多形核白细胞（PMN;中性粒细胞）从血流迁移和穿过膀胱上皮。故障解决感染在没有一个中性反应表明PMN在膀胱防御的重要性。以方便的膀胱上皮，肾盂肾炎大肠杆菌 （UPEC）定植，大多数尿路感染（尿路感染）的致病因子，抑制采用各种局部定义的机制的急性炎症反应。为了进一步调查主机和细菌病原体之间的相互作用，我们开发了UPEC先天免疫反应的这个方面的体外模型。在transuroepithelial嗜中性粒细胞迁移测定，在Boyden小室中的变化，培养的膀胱epithelial细​​胞生长至汇合的透气性支承底面。中性粒细胞是从人静脉血分离和被施加到膀胱上皮细胞层的基底一侧。中性粒细胞的迁移较响应于细菌感染或细胞趋化分子的生理学相关的基底外侧至顶端的方向被使用血细胞计数器计数。该模型可用于研究UPEC和真核细胞之间的相互作用以及由PMN来询问对膀胱上皮细胞的遍历的分子的要求。该transuroepithelial中性粒细胞迁移模式将进一步推动我们在膀胱初始炎症反应统一企业的理解。

该transuroepithelial中性粒细胞迁移实验使整个培养的膀胱上皮细胞层响应各种刺激（ 图1B）人类中性粒细胞迁移的定量评估。而该协议是简单的，也有许多变量，可以影响PMN的迁移，从而影响了该测定的可重复性。同时准备透水支持和中性粒细胞减少技术和生物学重复之间的变异应采取措施。例如，如果在一个实验中使用只含有足够汇合5637细胞层渗透性的支持。的5637细胞的汇合是使用...

Watch this Scientific Journal Video about Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium at JoVE.com

Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

人中性粒细胞迁移的跨越培养膀胱上皮定量评价

我们开发了一个体外模型，模仿与肾盂肾炎大肠杆菌膀胱感染，在急性炎症反应的重要组成部分。该transuroepithelial中性粒细胞迁移测定法使人类中性粒细胞迁移的定量评估跨越膀胱上皮，在可渗透载体培养中，响应于细菌感染或细胞趋化物质。

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. ...

Research

JoVE Journal

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

人外周血NK细胞的扩展，纯化和功能评估

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

科研

免疫与感染

Tracking Neutrophil Intraluminal Crawling, Transendothelial Migration and Chemotaxis in Tissue by Intravital Video Microscopy

追踪中性粒细胞腔内的爬行，活体视频显微镜Transendothelial组织中的迁移和趋化

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

细胞迁移的的Phagokinetic轨道运动MLCK的定量评价

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

体外 共生分析评估病原体诱导中性粒细胞跨上皮迁移

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Human Neutrophil Flow Chamber Adhesion Assay

人中性粒细胞流动室粘附实验

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

体外犬中性粒细胞胞外陷阱的形成: 荧光显微镜动态定量分析

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...