量<em在体外</em>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
Summary
中性粒细胞活化的内皮细胞在感染部位是坚持宿主的炎症反应的一个组成部分。本报告中所述的是中性粒细胞的结合测定法，它允许对<em>在体外</em>初级人类嗜中性粒细胞的定量静态条件下的炎症介质激活内皮细胞的结合。
Abstract
血管内皮细胞在炎症反应中起一个不可分割的一部分。在炎症的急性期，被激活的内皮细胞（ECs）的主机介质，或直接由保守的微生物成分或宿主来源的危险分子。活化的内皮细胞表达细胞因子，趋化因子和粘附分子，调动，激活和留住白细胞感染或受伤在现场。嗜中性粒细胞是白细胞到达，和坚持的内皮细胞上存在两种细胞表面粘附分子通过各种。嗜中性粒细胞的主要功能是直接消除微生物的威胁，促进招聘其他白细胞通过释放其他因素，并启动修复创面。因此，他们的招聘和附件的内皮细胞，是启动的炎症反应中的关键一步。在这份报告中，我们描述了一个在体外中性粒细胞粘附实验使用钙黄绿素AM标记初级人类嗜中性粒细胞在静态条件下，定量微血管内皮细胞活化的程度。此方法具有附加的优点，荧光分光光度法定量测定同一样品，也可以直接用荧光显微镜定性评估的中性粒细胞结合更可视化。
Introduction
由于血管内皮细胞在循环血液直接接触，它是唯一位于启动一个快速的在感染或损伤的炎症反应。内皮细胞（EC）表示模式识别受体，可以识别各种保守的细菌成分和危险分子，以及宿主的炎症介质如肿瘤坏死因子受体。这些受体的激活诱导内皮细胞分泌的细胞因子（ 例如 IL-6，IL-8，CXCL1和CCL2），并上调粘附分子（ 例如 E / P-选择素，VCAM-1和ICAM-1）在其细胞表面1 ，2。所有这些分子促进本地化的感染和损伤部位的白细胞，中性粒细胞对感染的反应，以明确的传染性病原体的主机并发起组织修复3,4涉及协调良好的血管内皮细胞和响应之间的相互作用中性粒细胞。 EC激活后，IL-8分泌，形成血管内梯度上的内皮细胞，使中性粒细胞感染或受伤的5,6家中到现场。 E / P-选择素介导的中性粒细胞捕获和通过相对较弱协会与glycomolecules中性粒细胞表面滚动。这些相互作用，以及与IL-8其同源受体结合，促进强劲，中性粒细胞在内皮细胞表面7-10整合素介导的附件，并最终逮捕。被逮捕后，嗜中性粒细胞可以迁移出具体感染部位，直接消除病原体，产生中性粒细胞胞外的陷阱，以防止病原体的传播，促进伤口愈合，并释放出额外的因素，招募其他的白细胞，如单核细胞，巨噬细胞和树突状血管细胞11-17。
本报告中描述的是一种体外的方法来定量中性粒细胞的粘附，术后1血管性内皮细胞活化后由主机炎症介质TNFα。本试剂盒的目的是评估的激活的内皮细胞，而不是中性粒细胞。初级人类嗜中性粒细胞是首先使用密度梯度分离分离，然后标记的乙酰氧基甲基钙黄绿素（AM）。酯酶的活细胞内水解钙黄绿素AM的高荧光钙黄绿素分子的激发492-495 nm，发射513-516纳米18。荧光标记的嗜中性粒细胞与EC的单分子膜，然后孵育，其后除去非粘附的中性粒细胞。剩余的，结合的嗜中性粒细胞的荧光，然后使用荧光分光光度计测定，计算作为一百分比的总的嗜中性粒细胞的荧光每孔输入。这种方法有绑定的钙黄绿素标记的嗜中性粒细胞用于分光额外的好处是可以用荧光显微镜直接观察，给予了更多的定性读出EC交流tivation。由于此法是在静态条件下，只有非常初步的事件发生在中性粒细胞粘附级联评审会。这也证实在本报告中，使用E-选择素抗体阻断，表明中性粒细胞粘附的TNFα处理人肺微血管EC（HMVEC龙）单层与E-选择素的相互作用时，大大减少被打乱。
除了对TNFα，我们已经成功地使用这种检测人脐静脉EC（HUVEC）激活Toll样受体1/2激动剂肽相关脂蛋白（PAL），胞壁质脂蛋白（MLP）和Pam3Cys和程度来确定龙HMVEC激活由Pam3Cys 19,20。此外，我们还成功地使用这种检测激酶抑制剂和RNAi介导的表面和胞质蛋白在HMVEC龙击倒后，表明这种方法是兼容与各种生化和screenin克分析20。综上所述，这个实验提供了一个易于使用的，重现性好，功能更强大的方式来访问EC活化程度的炎症介质在体外 。
Protocol
1。电镀和维护微血管内皮细胞解冻和增加你的微血管内皮细胞，根据制造商提供的说明。在这个协议中，细胞生长在EGM-2 MV媒体（龙沙），并建议所有的实验进行两周内解冻，以尽量减少由于通道数量的任何变化。我们的实验与HMVEC龙进行4至9之间的通道。 第1天：当细胞达到80-90％汇合，胰蛋白酶消化，悬浮在120,000细胞每毫升。板36,000细胞（0.3毫升），每孔48孔，聚苯乙烯组织培养…
Representative Results
为了获得可靠的，可再现的结果，使用中性粒细胞的结合测定，它是必不可少的健康和微血管内皮细胞的汇合天的测定是最优的HMVEC隆在图1中，如图所示。此外，它必须用于低通道数微血管内皮细胞（ 即小于9代），因此，我们建议所有的实验进行两周内解冻。嗜中性粒细胞的健康也是非常重要的，特别是因为钙黄绿素AM将不被代谢，如果细胞以某种方式由荧光钙黄绿素分子。?…
Discussion
对于一个成功的嗜中性粒细胞/微血管内皮细胞粘附试验的最重要的步骤是：1）使用低传代次数（<9），健康的内皮细胞; 2）维护分离出的嗜中性粒细胞的低密度（ 即 <5×10 6个细胞钙黄绿素AM标记的嗜中性粒细胞/毫升），并利用他们的隔离在两小时内，3）苛养洗涤，并使用钙黄绿素AM标记的嗜中性粒细胞中及时，以尽量减少EC从嗜中性粒细胞的钙黄绿素的污染和损失。分别。 </p…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是由加州大学旧金山分校的麻醉和围手术期护理部的支持。
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
| HMVEC-Lung | Lonza | CC-2527 | |
| EGM-2 MV | Lonza | CC-3202 | |
| HBSS | Life Technologies | 14175-095 | Can be substituted with any vendor |
| 48-well Tissue Culture Plates | BD Falcon | 353078 | |
| PBS (without phenol red) | UCSF Cell Culture Facility | CCFAL001 | Can be substituted with any vendor |
| D-PBS (without Ca2+ and Mg2+ and phenol red) | UCSF Cell Culture Facility | CCFAL003 | Can be substituted with any vendor |
| RPMI-1640 (without phenol red) | Life Technologies | 11835-030 | |
| Polymorphprep | Axis-Shield | 1114683 | |
| calcein AM | Life Technologies | C3099 | (0.995 mM) stock soln |
| Trypan Blue Solution | Sigma-Aldrich | T8154 | |
| 40 μM filters | VWR | 21008-949 | Can be substituted with any vendor |
| FLUOstar OPTIMA Spectrophotometer | BMG LABTECH | – |
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Citar este artigo
Wilhelmsen, K., Farrar, K., Hellman, J. Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions. J. Vis. Exp. (78), e50677, doi:10.3791/50677 (2013).