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。

1。电镀和维护微血管内皮细胞<ol><li>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。 </li><li>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，聚苯乙烯组织培养板（S）。包括HMVEC井， 不会与嗜中性粒细胞进行培养，以获得背景荧光读数。除非使用专门设计的荧光的检测方法，交替的行或列的48孔板，将最大限度地减少任何光线从邻近井的污染。注：井可以是预先涂有聚LYSINE，胶原蛋白或纤维连接蛋白。 </li><li>第2天：加入新鲜的培养基。 </li><li>第3天：相衬显微镜，确认细胞是健康的（ 即 “鹅卵石”外观和小细胞碎片），他们是100％的融合（ 图1）。如果细胞是不是100％融合，改变媒体的每一天，直到他们准备好了。 </li><li>一旦HMVEC单层准备治疗，洗一次细胞与汉克的平衡盐溶液（HBSS），加0.3毫升新鲜EGM-2 MV的媒体或媒体到合适的井含炎症激动剂。在这个协议中的HMVEC龙治疗与TNFα[100毫微克/毫升（PeproTech公司，新泽西州）3小时，因为这是一个很好的描述激活ECS 21。注：建议您的时间让您的活化HMVEC细胞（步骤4.1）和钙黄绿素AM实验标记的嗜中性粒细胞（步骤3.5），同时准备使用。我们需要约2.5小时到隔离和标签neutroph的ILS。 </li></ol> 2。使用Polymorphprep的中性粒细胞隔离<ol><li>隔离如前所述22，中性粒细胞，或用一个现成的密度梯度溶液从全血中分离中性粒细胞。用人Polymorphprep协议由Nuzzi 等人 2007年业绩的收益率约为2-3×10 6中性粒细胞每毫升全血23。注：为了避免过多的红细胞裂解，葡聚糖沉淀去除红血细胞之前，可以进行密度梯度离心24。 </li><li>带来Polymorphprep密度梯度溶液RT。 </li><li>收集30毫升的全血中的存在下，从健康人志愿者的抗凝血剂如肝素，柠檬酸盐或乙二胺四乙酸。的血应该在2个小时之内使用，并保持在18-22℃。 </li><li>层5毫升的全血在5毫升15毫升锥形管中的密度梯度溶液（图2A）。避免改变血液体积和密度梯度溶液，因为这可能会改变后续的离心条件。 </li><li>离心450×g离心30分钟，在18-22℃。确保慢慢加快离心机（ 即关闭离心机制动），以及平衡管，以防止振动，以帮助减少嗜中性粒细胞的激活，并防止层的混合。停止离心时间或更高的离心力将导致在较低的白细胞层，其中包含嗜中性粒细胞，，进一步迁移下来，对颗粒的红血细胞。 </li><li>吸出血浆和上部含白细胞频带的外周血单个核细胞层含有嗜中性粒细胞（ 图2B），以防止污染。 </li><li>使用1-2毫升血清吸管，删除含有嗜中性粒细胞的白细胞较低层。结合嗜中性粒细胞层成30毫升的PBS（不含Ca 2 +和Mg 2 +）在50毫升锥形管。 </li><li>使体积至50ml，用PBS（不含Ca 2 +和Mg 2 +），以450×g离心10分钟，在18-22℃。 </li></ol> 注：2.9和2.10步是可选的，但强烈推荐。 <ol start="9"><li>吸出上清液。中性粒细胞在8毫升无菌水重悬，持续30秒，以裂解红血细胞，立即加入2ml的5倍的PBS（不含Ca 2 +和Mg 2 +），用手轻轻混匀。 请勿培养细胞中的细胞悬液，以水时间超过30秒，因为显着的中性粒细胞可发生死亡。 </li><li>在250 XG离心5分钟，在18-22°C。 </li><li>液洗涤细胞一次，用10毫升的PBS（不含Ca 2 +和Mg 2 +）和在250×g离心5分钟，在18-22℃下</li><li>重悬在10ml的RPMI-1640（不含酚红）的嗜中性粒细胞，并使用台盼蓝计数活细胞染色排除法。大于5×10 6个每毫升的细胞密度，避免长时间，因为这可能会导致嗜中性粒细胞的激活。 </li></ol> 3。中性粒细胞贴标钙黄绿素AM <ol><li> 6×10 5个中性粒细胞的48孔板每孔使用。 </li><li>计数后，转移到50毫升锥形管，将重新悬浮的细胞和嗜中性粒细胞稀释至2-4×10 6细胞每毫升用无酚红的RPMI-1640。 </li><li>添加钙黄绿素AM到3微米（ 即 2.98微升钙黄绿素AM股票（1毫克/毫升），每毫升媒体）原液，轻弹管拌匀。大于5μM钙黄绿素AM将导致泄漏由嗜中性粒细胞。 注意：非荧光钙黄绿素AM被裂解的细胞内的内源性酯酶产生高荧光分子的钙黄绿素（ 即死亡的细胞，因此无法荧光）。 </li><li>用铝箔和孵化FO量管R 30分钟，在37℃下在黑暗中。 注：较长的孵育时间可能导致更多的钙黄绿素从嗜中性粒细胞的粘附实验的过程中解放出来。 </li><li>在450×g离心5分钟，在18-22℃下离心，洗涤用10毫升的RPMI-1640（不含酚红;预温至37℃）的嗜中性粒细胞2倍。第二次洗涤后，重悬细胞，首先通过嗜中性粒细胞，但40微米的无菌过滤器，以去除团块，然后一次以450×g离心5分钟，在18-22℃。 </li><li>重悬中性粒细胞在RPMI 1640（不含酚红;预热至37℃）在2×10 6嗜中性粒细胞每毫升。 </li></ol> 4。中性粒细胞结合分析<ol><li>洗净HMVEC单分子膜，用含3％BSA以每孔0.5ml的过滤灭菌（0.22微米）的RPMI 1640（不含酚红）的3倍。 </li><li>吸媒体，并添加6×10 5钙黄绿素标记的嗜中性粒细胞（0.3 ml的细胞悬液），或0.3毫升的RPMI 1640（不含酚红）无中性粒细胞，在合适的孔48孔板注意：这是一个相当于8×10 5个每1cm 2的中性粒细胞。 </li><li>孵育20分钟，在37℃，5％CO 2培养箱中 。 </li><li> 共有中性粒细胞的荧光（PRE洗涤）：测量各孔的荧光强度为485 nm的激发波长和发射波长为520nm（荧光集）滤波器在FLUOstar或等效，分光光度计。只执行该步骤之前，的孵化终点。 注意：在这个协议中，所发出的光的钙黄绿素的激发和检测进行从露天井的顶部，但是，两者也可以从孔的底部进行。 </li><li>洗净的孔中HMVEC单层和的5倍的嗜中性粒细胞用0.5毫升每孔的PBS（瓦特/的Ca 2 +和Mg 2 +）。卸下介质和清洗由反相板，并轻轻拍打到纸巾上，除去残留的液体。 </li><li>添加0.3毫升的RPMI 1640（不含酚红）每口井。 </li><li> 贴壁的中性粒细胞的荧光（柱洗）：在步骤4.4中，测量各孔的荧光强度。 </li><li>确定的百分比的坚持和相对坚持每口井： </li></ol> 注意：在这个阶段，可以通过以下方式获得的钙黄绿素标记的嗜中性粒细胞的图像，通过使用荧光显微镜能够配备标准的荧光过滤器。 图4中的图像上得到了奥林巴斯IX51倒置显微镜配备一个的Retiga 2000R相机（Q成像）使用Q-捕捉PRO7软件（Q成像）。

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+human+vascular+endothelial+cells+acquire+adhesiveness+for+neutrophils+after+stimulation+with+interleukin+1,+endotoxin,+and+tumor-promoting+phorbol+diesters.">Cultured human vascular endothelial cells acquire adhesiveness for neutrophils after stimulation with interleukin 1, endotoxin, and tumor-promoting phorbol diesters.</a> J. Immunol. 136, 649-654 (1986).</li><li>Gamble, J. R., Harlan, J. M., Klebanoff, S. J., Vadas, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+the+adherence+of+neutrophils+to+umbilical+vein+endothelium+by+human+recombinant+tumor+necrosis+factor.">Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 8667-8671 (1985).</li><li>Braut-Boucher, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178, 41-51 (1995).</li><li>Ait-Oufella, H., Maury, E., Lehoux, S., Guidet, B., Offenstadt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelium:+physiological+functions+and+role+in+microcirculatory+failure+during+severe+sepsis.">The endothelium: physiological functions and role in microcirculatory failure during severe sepsis.</a> Intensive Care Medicine. 36, 1286-1298 (2010).</li><li>Andonegui, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium-derived+Toll-like+receptor-4+is+the+key+molecule+in+LPS-induced+neutrophil+sequestration+into+lungs.">Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.</a> The Journal of Clinical Investigation. 111, 1011-1020 (2003).</li><li>Sharma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+endothelial+cell+platelet-activating+factor+production+and+inflammatory+cell+adherence+are+increased+in+response+to+cigarette+smoke+component+exposure.">Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 302, L47-L55 (2012).</li><li>Deban, L., Correale, C., Vetrano, S., Malesci, A., Danese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pathogenic+roles+of+microvasculature+in+inflammatory+bowel+disease:+a+Jack+of+all+trades.">Multiple pathogenic roles of microvasculature in inflammatory bowel disease: a Jack of all trades.</a> The American Journal of Pathology. 172, 1457-1466 (2008).</li><li>Gross, W. L., Trabandt, A., Csernok, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Wegener&amp;#39;s+granulomatosis.">Pathogenesis of Wegener&amp;#39;s granulomatosis.</a> Annales de Medecine Interne. 149, 280-286 (1998).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+inflammatory+cell+involvement+in+brain+arteriovenous+malformations.">Evidence of inflammatory cell involvement in brain arteriovenous malformations.</a> Neurosurgery. 62, 1340-1349 (2008).</li><li>Martens, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptides+which+bind+to+E-selectin+and+block+neutrophil+adhesion.">Peptides which bind to E-selectin and block neutrophil adhesion.</a> J. Biol. Chem. 270, 21129-21136 (1995).</li></ol>

作者什么都没有透露。

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（&lt;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 &lt;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...

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tivati​​on。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 。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>HMVEC-Lung</td>
 <td>Lonza</td>
 <td>CC-2527</td>
 <td></td>
 </tr>
 <tr>
 <td>EGM-2 MV</td>
 <td>Lonza</td>
 <td>CC-3202</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Life Technologies</td>
 <td>14175-095</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>48-well Tissue Culture Plates</td>
 <td>BD Falcon</td>
 <td>353078</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS (without phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL001 </td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>D-PBS (without Ca2+ and Mg2+ and phenol red)</td>
 <td>UCSF Cell Culture Facility</td>
 <td>CCFAL003</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>RPMI-1640 (without phenol red)</td>
 <td>Life Technologies</td>
 <td>11835-030</td>
 <td></td>
 </tr>
 <tr>
 <td>Polymorphprep</td>
 <td>Axis-Shield</td>
 <td>1114683</td>
 <td></td>
 </tr>
 <tr>
 <td>calcein AM</td>
 <td>Life Technologies</td>
 <td>C3099</td>
 <td>(0.995 mM) stock soln</td>
 </tr>
 <tr>
 <td>Trypan Blue Solution</td>
 <td>Sigma-Aldrich</td>
 <td>T8154</td>
 <td></td>
 </tr>
 <tr>
 <td>40 &mu;M filters</td>
 <td>VWR</td>
 <td>21008-949</td>
 <td>Can be substituted with any vendor</td>
 </tr>
 <tr>
 <td>FLUOstar OPTIMA Spectrophotometer</td>
 <td>BMG LABTECH</td>
 <td>-</td>
 <td></td>
 </tr>
 </tbody>
</table>

quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions

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。

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。?...

Watch this Scientific Journal Video about Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions at JoVE.com

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

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&gt;在体外</em&gt;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。

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 ...

quantitative-vitro-assay-to-measure-neutrophil-adhesion-to-activated

Research

JoVE Journal

Immunology and Infection

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

17.0K 次观看.  University of California, San Francisco. 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，它允许对在体外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。

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

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

到哺乳动物的上皮细胞在体外细菌粘附的含量

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

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, ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

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 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

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 ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

静电粘附实验对整合素激活的T细胞研究

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

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

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

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 ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

在体外和体内模型来研究细菌粘附于血管壁下流动条件

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...