The authors thank Dr. P.L. Hordijk for critically reading the manuscript. AED is supported by a Landsteiner Foundation for Blood Transfusion Research (LSBR) fellowship (grant #1028). JK is supported by the Dutch Heart Foundation (2005T3901). JDvB is a NHS Dekker fellow (grant #2005T039).

嗜中性粒细胞是从谁已经签署了知情同意书健康志愿者隔离。研究符合已执行与人类福利的机构和国家的指导方针。 1，电镀和人脐静脉内皮细胞的维护<ol><li>根据制造商的说明培养人脐静脉内皮细胞（HUVEC）。成长的HUVECs在纤连蛋白（FN）包被使用媒体菜（10微克/毫升，溶解于去离子水中）（内皮基础培养基（EBM-2）培养基补充有内皮细胞生长培养基（EGM-2）singlequots）。使用的细胞培养4-8代之间进行的实验。 </li><li>第1天：外套流室用100μl纤连蛋白（10微克/ ml，在PBS中）至少2小时，在37℃和5％CO 2。 </li><li>第2天：当细胞达到80-90％汇合，trypsinize经过仔细清洗，用RT磷酸盐缓冲液中的细胞（PBS）的pH值为7.4，离心800 XG，重悬在利用媒体80万个细胞/ ml。板80,000个细胞中的FN涂覆流动腔室的每一个信道，并轻轻吸取细胞悬液上下。文化的O / N的孵化器在37℃和5％CO 2。 </li><li>第3天：轻轻倾斜滑动以45°角刷新在流动腔滑媒体。 （参见图1A）。 注：建议仅移除在水塘，而不是在信道本身的媒体。除去介质中的信道可能导致血管内皮细胞的损失和死亡而引起的移液介质的拖力。 </li><li>检查用相差显微镜，如果内皮细胞形成的单层（ 即，100％汇合）。如果细胞是不是100％汇合时，每天更换介质两次，直至达到100％汇合。 </li><li>一旦细胞达到汇合，刺激细胞用培养基（见步骤1.1）包含炎症介质（TNF-α（10毫微克/毫升））。刺激的HUVECs O 2 / N（ 即，12小时）与TNF-α的结果，在血管内皮细胞的炎性表型， 即 ，上调细胞粘附分子如ICAM-1和VCAM-1的13的。 </li></ol> 2，多形核白细胞分离使用珀渐变<ol><li>分离多形核白细胞（PMN）之前：N-2-羟乙基哌嗪-N&#39;-2-乙磺酸（HEPES）-buffer（流缓冲器以下简称）制备。流缓冲器用于洗涤分离的多形核白细胞和作为缓冲在流化验。要准备流缓冲区的稀释：7.72克氯化钠（132毫米），4.76克HEPES（20毫米），0.45克氯化钾（6毫米），0.25克硫酸镁4•7H 2 O（1毫米），K 2 HPO 4•3H 2 O（1.2毫摩尔）在1升去离子水中，调节至pH为7.4（这股票可以保持在4℃下持续数周）。 </li><li>加入新鲜的100微升的1M 氯化钙＆＃160（1毫摩尔），2.5毫升从200g / L的储备液浓度（0.5％体积/体积）和0.1克葡萄糖至100毫升人白蛋白（0.1％重量/体积）的流缓冲器。接着，使用0.45μm的过滤器过滤流缓冲器。注：此流缓冲器需要准备新鲜的每一个实验。 </li><li>之前PMN的隔离，制备10％的柠檬酸三钠（TNC）的PBS，pH 7.4的溶液。 </li><li>收集20毫升全血中钠肝素vacuettes从健康志愿者。稀释全血1：1与10％的PBS /跨国公司在50ml试管和吸管20毫升仔细稀释血液到12.5毫升珀（一23％（W / W）在水中的胶体溶液与1.130克/毫升的密度）在一个新的50ml管中。小心地放置在离心机试管和自旋20分钟，在800 XG具有低加速度并且没有中断设定为室温。 注意：当添加的稀释血液的珀可，倾斜的含珀管在45°角，并轻轻吸取稀释血液在管机智小时最低吸管男孩的设置。 </li><li>除去所有液体，随后填充用冰冷的红细胞裂解缓冲液（4.15克氯化铵 [0.155 M]，0.5g的KHCO 3 [0.01 M]和18.5毫克EDTA（三重Ⅲ）[0.1毫米]至500ml的管冰冷的H 2 O）溶解红细胞。离开管在冰上，偶尔颠倒试管，直至悬浮液变成深红色，然后离心500×g离心5分钟，在4℃下以使能中断。 注：沉淀级分中含有的中性粒细胞（嗜中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞）与红细胞一起。 </li><li>除去上清液，并两次在冰冷的裂解缓冲液，在500 XG在4℃下洗涤沉淀5分钟。 </li><li>悬浮与流动缓冲颗粒在室温，并确定使用一个血球或自动细胞计数中性粒细胞浓度。暂停在流缓冲器中的中性粒细胞以1×10 6个细胞/ ml，并保持在室温。 </li></ol> 3，标签内皮枢纽人VE-cadherin和PECAM-1 <ol><li>添加粘附分子的Alexa Fluor 647抗体（克隆WM59）以1:100稀释和钙无关的VE-cadherin蛋白-FITC抗体（克隆55-7H1）在1:50稀释内皮细胞培养基（见步骤1.1）和开始流动实验期间的生理流动条件下的中性粒细胞轮回形象化内皮细胞的交界动力学之前温育30分钟。 注意：在添加直接标记的抗体的流动室中，确保在每个通道中的量不超过100微升。这有助于保持尽可能低的抗体费用。 </li></ol> 4，中性粒细胞透射电镜流分析<ol><li>将中性粒细胞进入水浴15分钟，在37℃下它们注入流动系统之前。 </li><li>管连接到一个空的流动腔室和填充用温水（ 例如 37℃）流动缓冲液（参见步骤4.3），以防止形成气泡设置日时ê流系统。 </li><li>连接的空流室向注射器泵流系统的一侧通过使用含有20ml的注射器的硅橡胶管，并把流腔进入显微镜的阶段。 </li><li>一个空的流动腔室的另一侧连接到充满37℃，流缓冲器（ 图1B）的贮存器烧瓶中，并开始在注射器泵中，以填补所有同流缓冲管。泵将拉从贮存在流缓冲器通过流腔进入注射器。注意：此管还包含一个串联的Luer注入口，它允许多形核白细胞以用针插入正在运行的实验被注入，而不停止流并产生气泡。 </li><li>更换空流室含TNF-α治疗内皮细胞流动室，连接载流缓冲管，并将其放置到显微镜载物台（ 图1C）。断开并重新连接之前夹断管米到含有内皮细胞，如不夹断腔，可能导致形成气泡的管子和/或流动腔室的内部。 </li><li>调整流速为1达因/厘米2，按照在后毛细血管小静脉与生理流速（1-5达因/厘米2）。 </li><li>记录微分干涉对比（DIC），FITC（488纳米）和Alexa Fluor 647（647纳米）使用共聚焦激光扫描显微镜同时。 </li><li>注入的中性粒细胞通过在网上的Luer注入口（ 图1D）用1ml注射器（步骤4.1）慢慢地在流动系统。 </li><li>几分钟后，出现白细胞，坚持和轮回。通过断开从流动室和移液固定液（在PBS中的3.7％甲醛）的管道到流动室停止实验，在任何需要的时刻。允许固定10分钟，然后用PBS洗涤。数据是使用图像处理软件（见材料和​​设备表）进行分析。 注：执行使用在37℃，5％CO2配备了气候室具有恒定 ​​温度的共聚焦激光扫描显微镜和63X，他的实验油目标。 </li></ol>

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The authors have no competing financial interests.

对于此协议，它是重要的，以防止气泡在流动室的形成，因为这会导致细胞凋亡和破坏的单层。为了避免这种情况，我们想强调，要特别关注的步骤4.2和4.5，其中所述管被连接到流动室。在该协议的另一个重要步骤是，保持在室温下通过它们注入到流动室（步骤4.1）之前培养他们15-30分钟，在37℃的中性粒细胞的灌注。这导致白细胞整合素涂刷，允许他们坚持粘附分子如ICAM-1或VCAM-1的内皮细胞。 这个协议并不限于研究嗜中性粒细胞的轮回。还其他白细胞的类型，如单核细胞或淋巴细胞可被使用。注意，步骤4.1，也就是说 ，吸白细胞的白细胞类型之间可能有所不同。也可以使用其它类型的内皮细胞。对于这一点，仍然是关键刺激内皮细胞与适当的炎症刺激，如TNF-α或IL-1β。若嗜中性粒细胞中transmigrating不响应，可以考虑处理的嗜中性粒细胞短时间（5分钟），用N-甲酰基-L-蛋氨酰-L-亮氨酰-L-苯丙氨酸（fMLP的）肽16。这会刺激嗜中性粒细胞，特别是它们整合，甚至进一步，使它们更容易粘附到内皮。 通常为1达因/厘米2流速使用。这种流动的速度是衡量后毛细血管小静脉，大多数白细胞跨内皮迁移发生17个 。使用所描述的流动室中，所以能够提高流速高达10达因/厘米2。然而，不推荐使用，以增加抗剪进一步。它可能会导致不希望的泄漏从流动室的细胞的管道和脱离。 在图3中所描述的结果表明，这些抗体可用于可视化和中白细胞血细胞渗出研究内皮细胞 - 细胞连接的动态。特别是，除了现有的轮回测定此协议允许的生理流动条件下在实时下以鉴别从跨细胞迁移的细胞旁。由于抗体染色的细胞 - 细胞连接，可以比分白细胞跨越VE-钙粘着蛋白/ PECAM-1-阳性结与非正VE-钙粘着蛋白/ PECAM-1位点的数目。通过这种方式，跨细胞的迁移可以从细胞旁迁移来区别。 的PECAM-1抗体用于本研究是针对第二胞外域：它要强调，这些抗体不与它们结合到蛋白质的功能干扰是很重要的。铺板在该抗体的存在下在没有显示在扩频或形成一单分子层的任何缺陷内皮细胞，这表明该抗体至少没干扰内皮细胞之间的同型相互作用。在除此之外，两种抗体不损害中性粒细胞的迁移穿过内皮细胞单层的能力。此外，嗜中性粒细胞的跨内皮单层轮回在不存在或该抗体的存在的数量不改变（数据未显示）。重要的是，激光扫描共聚焦显微镜让我们能够同时录制不同的荧光通道和DIC的可能性。 因此，该测定法允许学习VE-钙粘蛋白和PECAM-1的动力学同时当嗜中性粒细胞穿过内皮细胞-细胞连接，并将有助于理解为什么白细胞选择一个路线上的其他， 即旁与跨细胞。

效率和严格控制的白细胞跨内皮迁移（TEM）是在生理过程，如免疫监视和急性炎症的关键重要性。然而，在某些病理生理条件下，不受控制的和过度的TEM观察所得的慢性炎性疾病（ 如类风湿关节炎，动脉粥样硬化，哮喘）。另外，在肿瘤细胞的转移，跨内皮迁移的过程是有助于对肿瘤细胞离开循环转移1-3。为了专门与过度白细胞或肿瘤细胞的TEM干扰，这个过程的调节有详细的了解是必要的。 据认为，在TEM过程是通过不同的步骤。开创性研究，回顾在二十年前由屠夫和施普林格，导致了多级模型描述TEM 4,5的过程。这种模式仍然适用，虽然有些广告ditional措施已列入6。阿龙等人描述的需要固定的趋化因子的内皮7的表面上的存在。最近，他们发现，血管内皮细胞本身产生这在内皮顶面8呈现的趋化因子。此外，同组提出的流动条件的重要性TEM 7时。近日，专注于两个不同的路线白细胞一些出版物可以在透射电子显微镜的最后血细胞渗出阶段。他们可以通过细胞间连接处， 即旁迁徙路线，或穿越内皮细胞体，被称为跨细胞迁徙路线9。卡曼和他的同事研究了这些途径中的细节和结论是白细胞穿越的脐静脉内皮细胞单层10时优先选择旁迁徙路线（90％），在跨细胞途径（10％）。然而，当被用来从其他来源的内皮细胞， 例如，脑或微脉管系统，更白细胞用的跨细胞途径（30％）11。该Vestweber组最近发现，当细胞-细胞连接处无法彼此通过使用敲除动物模型，替换内源的离解VE-钙粘蛋白的VE-钙粘蛋白-α-连环蛋白嵌合体，白细胞TEM被完全阻断12 。令人惊奇的是，作者发现，TEM被挡在数个，但不是所有的组织。总体来看，这些典雅的实验表明，白细胞优于跨细胞途径的路线旁，虽然触发此决定的调控信号还是一个未知数。 尽管大多数白细胞喜欢旁迁移路线，但仍难以既通路之间进行区分。除此之外，尽管许多研究集中在内皮细胞 - 细胞junct的作用离子，这些路口的动态，特别是连接蛋白VE-cadherin和PECAM-1，在此期间白细胞道口仍在争论。我们开发了一种相对简单的测定，其中这些结分子可在实时过程中使用荧光标记的抗体的生理流动条件下，白细胞血细胞渗出来监测。这些抗体不干扰或阻止目标蛋白质的交界完整或移动性。此测定法使得我们旁TEM的过程中跟随连接蛋白的动力学。此外，该法还允许歧视性的对位和跨细胞迁徙路线之间。

<table border="0" cellpadding="0" cellspacing="0" width="1251">
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			<td height="21" style="height:21px;width:471px;">Name of Material/ Equipment</td>
			<td style="width:365px;">Company</td>
			<td style="width:184px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td style="width:184px;">80606</td>
			<td>Flow-chamber</td>
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			<td height="20" style="height:20px;">0.45 &#956;m filter&#160;</td>
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			<td style="width:184px;">10462100</td>
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			<td height="20" style="height:20px;width:471px;">1 mL syringe&#160;</td>
			<td style="width:365px;">BD Plastipak</td>
			<td style="width:184px;">300013</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">20 mL syringe&#160;</td>
			<td>BD Discardit II</td>
			<td style="width:184px;">366296</td>
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			<td height="20" style="height:20px;width:471px;">21G needle&#160;</td>
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			<td style="width:184px;">301155</td>
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			<td height="20" style="height:20px;width:471px;">Ammunium chloride (NH4Cl)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1009245000</td>
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			<td height="26" style="height:26px;width:471px;">Calcium chloride (CaCl2)</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">449709</td>
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			<td height="40" style="height:40px;width:471px;">EBM-2 Basal medium + EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
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			<td>media</td>
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			<td style="width:184px;">1370041000</td>
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			<td style="width:365px;">Sigma-Aldrich</td>
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			<td height="20" style="height:20px;width:471px;">Glucose</td>
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			<td height="19" style="height:19px;">In-line Luer Injection port&#160;</td>
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			<td style="width:184px;">10820</td>
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			<td height="26" style="height:26px;width:471px;">Magnesium sulfate heptahydrate (MgSO4.7H2O)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">105886</td>
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			<td height="20" style="height:20px;">PECAM-1-ALEXA-647&#160;</td>
			<td style="width:365px;">BD Pharmingen</td>
			<td style="width:184px;">561654</td>
			<td>clone WM59 
			stock concentration 0.1mg/mL</td>
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			<td height="20" style="height:20px;">Percoll&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td style="width:184px;">17-0891-09</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:471px;">Phosphate Buffered Saline</td>
			<td style="width:365px;">Fresenius Kabi&#160; Nederland</td>
			<td style="width:184px;">M090001/01NL</td>
			<td>PBS</td>
			<td></td>
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			<td height="20" style="height:20px;width:471px;">Potassium chloride (KCl)</td>
			<td style="width:365px;">Sigma-Aldrich</td>
			<td style="width:184px;">P9541</td>
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			<td height="26" style="height:26px;width:471px;">Potassium hydrogen carbonate (KHCO3)</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1048540500</td>
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			<td height="26" style="height:26px;width:471px;">Potassium phosphate dibasic trihydrate (K2HPO4)</td>
			<td style="width:365px;">Sigma-Aldrich</td>
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			<td style="width:365px;">VWR</td>
			<td style="width:184px;">228-4331</td>
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			<td style="width:365px;">Calbiochem (Millipore)</td>
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			<td height="20" style="height:20px;">Syringe pump&#160;</td>
			<td style="width:365px;">Harvard Apparatus</td>
			<td>model number 55-5920</td>
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			<td height="20" style="height:20px;">TNF&#945;&#160;</td>
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			<td style="width:184px;">300-01A</td>
			<td>tumor necrosis factor</td>
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			<td height="20" style="height:20px;">trisodium citrate&#160;</td>
			<td style="width:365px;">Merck</td>
			<td style="width:184px;">1.06447.5000</td>
			<td>TNC</td>
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			<td height="20" style="height:20px;">Vacuettes</td>
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			<td style="width:184px;">560411</td>
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			<td></td>
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			<td height="20" style="height:20px;width:471px;">Zen Software 2008</td>
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</table>

real-time imaging of endothelial cell-cell junctions during neutrophil transmigration under physiological flow

在炎症过程中，白细胞离开流通和跨内皮打在下面的组织入侵的病原体。这个过程被称为白细胞跨内皮迁移。两条路线的白细胞穿过内皮细胞单层进行了说明：在旁路线， 即通过细胞-细胞连接和跨细胞途径， 即，通过内皮细胞体。然而，它在技术上难以对位和跨细胞途径区分。我们开发了一个简单的体外实验，研究内源性的分布VE-cadherin和PECAM-1的生理流动条件下，中性粒细胞跨内皮迁移过程中。在此之前灌注中性粒细胞，内皮细胞，短暂治疗对VE-cadherin和PECAM-1荧光标记的抗体。这些抗体不与这两个蛋白质的功能干扰，如由电小区substra确定TE阻抗传感和FRAP测量。使用这种测定法，我们能够遵循内源的分布VE-钙粘蛋白和PECAM-1跨内皮迁移中的流动条件下和在整个血管内皮细胞的白细胞的对位和跨细胞迁移路线之间进行区分。

我们首先测试了，如果抗体不与内皮细胞的屏障功能的干扰。我们通过使用电细胞 - 基底阻抗感测（ECIS）测定内皮细胞单层的电阻。有关详细信息，请参阅范Buul 等人 13时抗VE-钙粘蛋白的荧光标记的抗体加入到细胞中（ 图2A）中观察到的电阻不改变。抗VE-钙粘蛋白抗体，该抗体是公认的阻断VE-钙粘蛋白的功能降低的电阻显着（ 图2A）。另外，用于成像不改变的动力学的抗体VE-钙粘蛋白，如通过测定荧光恢复的光致漂白（FRAP）后评估VE-钙粘蛋白-GFP（ 图2B）。 1-2分钟之后，嗜中性粒细胞粘附在活化的内皮单层的可在DIC的信道（ 图3A）被可视化。爬行5-30 SE后C，嗜中性粒细胞中的绝大多数通过通过进行标记与抗PECAM-1的抗体和VE-钙粘蛋白的细胞 - 细胞连接的内皮细胞单层transmigrated。在血细胞渗出的过程中，分配VE-钙粘蛋白和PECAM-1，随后在实时（ 图3B）。在中性粒细胞白细胞渗出的部位，VE-cadherin蛋白在当地破坏，PECAM-1表现出了更多的环状结构。血细胞渗出完成后，关闭和路口VE-cadherin和PECAM-1显然搬迁的血细胞渗出的部位（ 图3C）。需要注意的是嗜中性粒细胞的表面上进行检测的抗PECAM-1抗体的部分，一旦嗜中性粒细胞达到内皮的BASO-侧面。然而，这并没有阻止中性粒细胞穿越血管内皮细胞。所观察到的动态VE-cadherin在实时中性粒细胞跨内皮迁移期间，在与肖和他的同事的工作协议谁结果显示，使用共聚焦显微镜和VE-钙粘蛋白-GFP转染的内皮细胞，该VE-钙粘蛋白-GFP扩散横向时白细胞越过内皮细胞间连接处14。工作也由苏和他的同事强调我们的PECAM-1的观察。他们对白细胞通道显示，旁边的横向VE-cadherin的扩散，PECAM-1在当地重新分配到各地的transmigrating中性粒细胞15环。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/51766/51766fig1highres.jpg" width="400" /> 图1 的体外流动室（A）箭头表示从该介质需要刷新的水库。 （B）的硅管，与用于注入中性粒细胞，无需断开管路（箭头）的直列鲁尔注射端口相连的流动室。 （ 三）康涅狄格州挠度的空流动室填充有37℃流动缓冲液（箭头）的贮存器的烧瓶中。流室（D）的连接用TNF-α处理的内皮细胞的含流缓冲管，并放置在显微镜载物台（箭头）。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51766/51766fig2highres.jpg" width="500" /> 图2：VE-钙粘蛋白抗体不与交界动力学或功能干扰：（A）内皮细胞单层阻抗是利用ECIS测量。 Y轴表示阻抗的欧姆和x轴表示时间（小时）。 VE-钙粘蛋白抗体克隆55-7H1，标有ALEXA647（蓝线）或同种型IgG的ALEXA647控制（红线）不改变内皮细胞单层阻抗，而VE-钙粘着蛋白阻断抗体CL75（黑线）并降低阻抗。 （二</stro纳克&gt;）VE-钙粘蛋白-GFP表达在血管内皮细胞，并使用共聚焦显微镜FRAP分析发现在荧光恢复没有变化的存在或不存在的VE-钙粘蛋白55-7H1抗体。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51766/51766fig3highres.jpg" width="500" /> 图3：VE-钙粘蛋白和PECAM-1分配过程中的实时嗜中性粒细胞的TEM（A）嗜中性粒细胞（标有白线）粘附于内皮细胞和交叉的细胞-细胞连接，而不会影响的分布VE-钙粘蛋白和PECAM -1。 （B）中的中性粒细胞穿越通过细胞与细胞间连接处的单层内皮细胞。的地方分散VE-钙粘蛋白和PECAM-1可以在嗜中性粒细胞突出通过细胞与细胞间连接处被观察到。白线示出嗜中性粒细胞的存在仍然对内皮的顶部。黄线显示​​中性粒细胞的膜塔t是已经内皮下。 （ 三）中性粒细胞已经完全越过了单层内皮细胞。 VE-cadherin和PECAM-1在搬迁的血细胞渗出的网站。黄线表示transmigrated中性粒细胞的边界。 <a href="https://www.jove.com/files/ftp_upload/51766/51766fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow at JoVE.com

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

白细胞交叉使用旁或跨细胞途径的单层内皮细胞​​。我们开发了一个简单的试验，以遵循内源交界的分布VE-cadherin和PECAM-1白细胞跨内皮迁移过程中的生理流动下两路轮回区分。

内皮细胞，细胞连接实时成像过程中中性粒细胞轮回在生理流动

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

real-time-imaging-endothelial-cell-cell-junctions-during-neutrophil

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Cell Biology

Immunology and Infection

Unit 1

Cell-Cell Interactions

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19.4K 次观看.  Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam. 白细胞交叉使用旁或跨细胞途径的单层内皮细胞​​。我们开发了一个简单的试验，以遵循内源交界的分布VE-cadherin和PECAM-1白细胞跨内皮迁移过程中的生理流动下两路轮回区分。 

视频: Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

白三烯B实时成像 4介导的细胞迁移和BLT1与β- arrestin的相互作用

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

科研

免疫与感染

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

分离人脐静脉内皮细胞和水流条件下其使用中的中性粒细胞轮回研究

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

T细胞实时成像实时信号复合物的形成

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

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 ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

用两相循环流系统实时成像和定量真菌生物膜的研究

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...