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的组织培养瓶。 </li><li>用5％CO 2孵育烧瓶中，在37℃，在湿润的气氛中，直到细胞的约95％汇合（〜6×10 6每烧瓶中的细胞），约4天。 </li><li>继代培养的5637细胞: <ol><li>从烧瓶中取出培养基。用10ml的Dulbecco磷酸盐缓冲萨林洗细胞E（DPBS）在室温下。 </li><li>加入6毫升温（37℃）0.05％胰蛋白酶，0.02％EDTA的溶液到烧瓶中，并置于37℃，5％CO 2中15分钟。 </li><li>通过离心将细胞转移到15毫升锥形管中，并沉淀在300×g离心5分钟。除去胰蛋白酶/ EDTA溶液。 </li><li>重悬细胞于6ml的RPMI +（10 6个细胞/毫升）中，并转移1毫升（10 6个细胞）到一个新的75 厘米烧瓶含20ml RPMI +。 </li></ol></li><li>重复步骤1.4继代培养细胞每4天或当细胞达到95％汇合。 </li></ol> 2。在透水支撑播种和成长5637细胞采用无菌技术进行了组织培养罩下面的步骤。为获得最佳效果，请使用经历少于10次文化5637细胞。 <ol><li>用无菌镊子，反转渗透支撑在无菌最深25 mm组织培养皿。 </li><lI&gt;胰蛋白酶消化75cm 2的烧瓶中加入5637细胞在95％汇合时，根据步骤1.4.1-1.4.3。 </li><li>使用1,000微升移液管，轻轻重悬细胞在约2ml的RPMI +至3×10 6个细胞/ ml时，通过细胞使用血球计数法测定其浓度。 </li><li>申请加入50μl细胞悬液（1.5×10 5个细胞）的各透气性支承而不接触该膜。放置在培养皿的盖子，然后小心地将培养皿在37℃，5％CO 2的时间不超过16小时。 </li><li>用无菌镊子，右可渗透支撑成24孔板含有0.6毫升RPMI +每孔。添加0.1毫升RPMI +每个透水支持上水库。在37℃，5％CO 2。 </li><li>更换培养基，每2天。第一，抽吸物从上部贮接着下部储介质，然后应用新鲜RPMI +中的顺序相反，在下部储存器（0.9毫升）中，然后在上RESErvoir（0.1毫升）中。 </li><li>无欲无求的渗透与支持5637个单元格后七天，评估细胞的汇合。用RPMI +（〜0.35毫升）补上水库。将细胞充分地汇合时，介质不会上部和下部油箱之间的平衡。 </li></ol> 3。细菌接种的制备<ol><li>使用无菌技术，加入20毫升适当的培养液，以250毫升的烧瓶用盖。使用LB培养基（LB）肉汤为E.大肠杆菌培养，并添加抗生素发酵液（如适用）。 </li><li>用无菌接种环，接种与从甘油菌或条纹板的肉汤。对于UPEC菌株，培养细菌培养，在37℃下约16小时，无晃动（促进生产型菌毛1）。 </li><li>转移的细菌培养到离心管中。通过离心沉淀细菌在8,000 xg离心10分钟。 </li><li>倒出上清液，重悬细菌在PBS至OD 600 = 1.000，相当于〜10 9 CFU /毫升。 <ol><li>以产生热杀死的细菌的刺激，温育的等分试样（ 例如，〜1.5×10 8 CFU在150微升）中重悬细菌在55℃下进行30分钟的。板式热灭活悬浮液的等分试样，以证实细菌死亡。 </li></ol></li><li>临用前，稀释悬浮细菌（直播或热灭活）10倍至10 8 CFU /毫升的温水无血清的RPMI在离心管[称为RPMI-]。 </li></ol> 4。从外周血分离人类中性粒细胞血液从成年志愿者集合需要事先审查和机构审查委员会的批准。穿戴合适的个人防护装备，并妥善处理有害物质，避免接触到人的血液。 <ol><li>大约25毫升的静脉血从一个健康的成年人volunteer应当由受过训练的人员放血被卷入3无菌钠含有肝素的血液收集管。 <ol><li>紧紧包住橡胶止血带绕上臂，肘部以上。 </li><li>消毒进入现场，肘窝，用消毒过70％的酒精擦拭。 </li><li>连接塑料管持有人有翅膀的蝴蝶针系统。 </li><li>针插入肘前静脉朝上30度角以内，斜角。针头刺破了静脉时，鲜血涌出般地出现在塑料管。 </li><li>插入采血管放入塑料管夹持器。当第一个收集管已满，请更换与第二个收集管。重复与第三管。 </li><li>当第三个收集管接近半满，取出止血带。 </li><li>覆盖穿刺部位用无菌棉球，慢慢地从静脉抽出针头。滑动保护罩在针和地点我NA生物危害锐器容器。 </li><li>将压力施加到穿刺部位。盖穿刺部位和棉花球用粘性绷带。 </li><li>轻轻颠倒采血管驱散肝素。 </li></ol></li></ol>采用无菌技术进行了组织培养罩下面的步骤。 <ol start="2"><li>用10毫升吸管，轻轻血液转移到一个新的，无菌的50ml锥形管中。添加3％的等效体积（w / v）的葡聚糖​​在0.9％NaCl和通过倒置混合。孵育管直立于室温下20分钟。 </li><li>而不破坏下层，小心吸出上层，并将其转移到一个新的50ml锥形管中。通过离心沉淀细胞，在300×g离心10分钟。弃上清。 </li><li>重悬细胞沉淀的体积的0.9％NaCl的相当于血液的起始体积。 </li><li>层下的细胞悬液，第10页毫升密度离心溶液贮留在两相之间的界面。离心机在400×g离心30分钟，不制动。弃上清。 </li><li>以裂解剩余的红血细胞，重悬细胞沉淀在10ml冷的0.2％NaCl中。温育30秒，然后迅速加入10毫升冷的1.6％NaCl溶液，以恢复等渗。 </li><li>通过离心沉淀细胞，在300×g离心6分钟。弃上清。 </li><li>重复步骤4.6-4.7，直到细胞沉淀似乎是自由的红血细胞，通常为3个回合的裂解。 </li><li>使用1,000微升移液管，重悬细胞沉淀，主要是中性粒细胞，在温热（37℃）的RPMI-至浓度为10 7个细胞/ ml时，通过使用血球细胞计数测定。保持所述细胞在37℃，直至使用。通常情况下，中性粒细胞活力和纯度为&gt; 99％为染色，分别评估后，台盼蓝染色的核形态和可视化。 </li></ol> 5。 Transuroepithelial中性粒细胞迁移作为说采用无菌技术进行了组织培养罩下面的步骤。仅使用透水支撑轴承5637融合细胞层，如步骤2.7确定。含有24孔板的RPMI-可以预先制备并保持在37℃，5％CO 2中，直到使用。 <ol><li>分装1毫升在24孔板预热的RPMI-每口井，准备每透水支持3口井。 </li><li>从可渗透支撑件的上部和下部油箱吸出培养基。 </li><li>用无菌镊子，可渗透载体转移到在步骤5.1中制备的24孔板中。通过传送支持从井洗井透水支持三倍。 </li><li>如果细菌接种物是被使用，颠倒可渗透支撑在无菌25毫米深层组织培养皿中。如果一个趋化因子（ 如 ，N-甲酰-Met的-LEU-PHE（FMLF）或IL-8）是被使用，继续进行步骤5.6。 </li><li>对于活的或死BACT实验里尔的刺激，每次接种透水支持的顶侧60μLRPMI-（模拟感染）或细菌接种（6×10 6 CFU）在步骤3.5准备。将盖子上的菜和孵化在37℃，5％CO2 1小时。 </li><li> RPMI-每孔加入0.6毫升到24孔低附着板，准备1井每透水支持。准备3口井含0.5毫升RPMI-枚举PMN输入。 <ol><li>如果一个趋化以代替细菌被使用时，趋化因子加到0.6毫升RPMI-在步骤5.6中制备的24孔板中。 </li></ol></li><li>右可渗透支撑到24孔低安装板。 </li><li>加0.1毫升PMN（10 6 PMN），在步骤4.9制得，各透气性支承的上部储器（基底侧）。加入100μlPMN直接到含有500μl的井RPMI-。在37℃，5％CO 2的1小时。 </li><li>用无菌镊子，轻轻刮去对井的边缘性支撑的膜从心尖部取出额外的中性粒细胞，然后弃置的支持。 </li><li>收集PMN通过轻轻刮取每个孔的底部与1,000微升移液管尖，并转移PMN悬浮液至微量离心管中。 </li><li>使用血球列举的PMN。 </li><li>计算中性粒细胞在较低水库的总数，由6000乘以中性粒细胞，以1 mm 2的数量（100 NL）。数据可以被报告为输入中性粒细胞百分比，或作为中性粒细胞数目归一化到10 6输入的PMN。 </li></ol>

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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

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。

人中性粒细胞迁移的跨越培养膀胱上皮定量评价

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. ...

quantitative-assessment-human-neutrophil-migration-across-cultured

Research

JoVE Journal

Immunology and Infection

15.0K 次观看.  Washington University School of Medicine. 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。 

视频: Quantitative Assessment of Human Neutrophil Migration Across a Cultured Bladder Epithelium

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

迁移人T淋巴细胞分离，培养和分析<em在体外</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

科研

免疫与感染

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

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

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 ...

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

The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation1,2. In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling3. Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)4. Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment2,5. In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm3 size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these neutrophil recruitment parameters can be stably determined and the single cell locomotion conveniently tracked in vivo.

We describe a protocol of brightfield intravital microscopy for measuring dynamic neutrophil-endothelial cell interactions during neutrophil recruitment in response to the source of a neutrophil chemoattractant in vivo. Neutrophil intraluminal crawling, transendothelial migration and chemotaxis in mouse cremaster muscle tissue are visualized with time-lapsed video photography and tracked with ImageJ.

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

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

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

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

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

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators 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

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

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

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

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

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

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 ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

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

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

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

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

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 response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

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

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence 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 ...