这项工作是由狼疮研究所（纽约州，纽约州），美国国立卫生研究院P01-AR49084，美国国立卫生研究院R21-DA026956和美国国立卫生研究院UL1-TR00165赞助。我们感谢罗伯特·P.金伯利博士为他的继续支持。

招募本研究所有捐助者签署知情同意书和研究经阿拉巴马大学伯明翰分校机构审查委员会。 1，血管内皮细胞初代培养和传代<ol><li>培养人脐静脉内皮细胞（HUVEC） 在体外在由血管内皮细胞基础培养基中的完全生长培养基（见材料清单），补充有内皮细胞生长因子。 注：在本研究中使用的生长因子包括：5毫微克/毫升的人重组表皮生长因子（hEGF的），1.0毫克/毫升氢化可的松，50毫克/毫升庆大霉素和50毫微克/毫升两性霉素-B（GA-1000），2个％胎牛血清（FBS），0.5纳克/毫升的血管内皮生长因子（VEGF），10纳克/毫升的人类成纤维细胞生长因子基本与肝素（hFGF-B），20纳克/毫升的重组人胰岛素样生长因子（ R 3-IGF-1），1微克/毫升抗坏血酸和22.5微克/毫升肝素。完全培养基制备最初在37℃和然后可被储存在4℃下1个月的制剂中使用。 </li><li>准备一个烧瓶中的细胞，完全培养基加入到75 厘米的组织培养烧瓶中（1毫升/ 5厘米2），然后将烧瓶使其平衡至37℃/ 5％CO 2的加湿培养箱中培养至少30分钟。人脐静脉内皮细胞将被直接接种到这个平衡的培养烧瓶中，在2,500-5,000细胞/ cm 2的密度。 </li><li>虽然媒体是平衡，迅速解冻股票脐静脉内皮细胞冻存管在37℃水浴。通过涡旋使细胞分散在原始储存小瓶，然后将它们直接添加到含有预平衡的HUVEC完全生长培养基中以达到2500-5000个细胞/ cm 2的密度培养瓶中。轻轻摇动烧瓶以均匀分布的细胞，然后将烧瓶返回到培养箱中。这些细胞现在代表的第一通道。 </li> &lt;李&gt;中应改为每两天，直到细胞70-80％汇合。 </li><li>在内皮细胞，现在可以从培养瓶中用胰蛋白酶/ EDTA收获。培养基从培养瓶中后跟的PBS冲洗，以从细胞中除去任何残留的蛋白和钙吸出。 0.25％胰蛋白酶-EDTA溶液，并在2-6分钟细胞脱离所评估光镜应该是显而易见的。 </li><li>当90％的细胞被四舍五入掉板中，加入2倍的胰蛋白酶抑制剂的等体积停止胰蛋白酶消化。为了便于细胞的收获，加入完全生长培养基的另一个等量。转移分离的细胞中的无菌15ml离心管中。 </li><li>离心分离的细胞在225×g离心5分钟，在室温下进行。吸出上清液，然后重悬细胞在2-3完全生长培养基中。测定细胞浓度和存活率用血球计数器和台盼蓝。 </li><li>利用日Ë收获细胞进行研究，重新播种额外75 厘米瓶与细胞以10,000细胞/ cm 2的密度，并继续执行步骤2.2。或者，细胞可以在此时被冻结为今后的研究。用于细胞冷冻，离心沉淀细胞，在225×g离心5分钟，在室温下进行。吸出上清，重悬细胞沉淀在含胎牛血清10％DMSO中以1×10 6个细胞/ ml的浓度。在细胞悬浮液中的细胞在细胞冷冻集装箱冷冻后，然后转移到冷冻管并储存于-80℃。 </li></ol> 2，血管内皮细胞层的制备<ol><li>利用冷冻第二通道脐静脉内皮细胞：复兴与文化。如果使用生长活跃的细胞，进行步骤2.2。 <ol><li>含解冻迅速从1.8步在37℃水浴2 次通过脐静脉内皮细胞的离心管。从离心管转移细胞到15ml的无菌离心管中，并加入10 ml生长的Medi嗯。 </li><li>离心细胞，在200×g离心5分钟，在室温下和吸出上清液，以除去残留的DMSO。 </li><li>重悬细胞沉淀用完全生长培养基中，将细胞转移至75 厘米的烧瓶中。加生长培养基至总体积为约20-25毫升，孵育37℃/ 5％CO 2在。 </li></ol></li><li>每天目视检查的汇合细胞培养与媒体的变化，每两天。一般于2-4天，电池将达到80-90％汇合，届时他们将准备在流室法的使用。 </li><li>要准备培养皿，将在流室中使用（见第5章），加入1毫升10微克/毫升纤维连接蛋白和0.05％（W / V）明胶为35 mm组织培养皿和吸管数次，使确保整个板表面涂。除去多余的纤连蛋白和明胶溶液和空气干燥该板为至少30分钟，以优化蛋白基质的形成。该fibronectin和明胶溶液可以重复使用高达10倍。 </li><li>按照步骤1.5-1.7描述使用胰蛋白酶-EDTA从步骤2.2收获HUVEC细胞。种子500,000细胞到每个包被的组织培养皿中。加入2ml生长培养基中每道菜，并培育37℃/ 5％CO 2的。 </li><li>使细胞生长至汇合，如步骤2.2。细胞应进行外观与媒介改变了每两天每天检查。在此之前的流室法，首要的HUVEC与20毫微克/毫升的人TNF-α的4-6小时上调，刺激黏附分子的表达。 </li></ol> 3，纯化配体涂层<ol><li>吸取0.5厘米直径的一个圆在35毫米组织培养皿的中心标记或钢笔。 </li><li>板20微升的标志区20微克/毫升的蛋白A。用枪头传播蛋白A的解决方案，以支付0.5厘米直径的圆内的整个区域。但千万不要触摸或刮伤D的表面是很重要的十岁上下。在37℃下1小时。 </li><li>用1毫升的PBS（pH 8.0）中洗蛋白A涂板3次。 </li><li>板加入50μl的1％BSA中的显着区域来阻止在板的非特异性结合。孵育2小时，在4℃下</li><li>用1毫升的PBS中，如步骤3.3洗阻断板3次。 </li><li>准备的Fc粘附受体配体嵌合蛋白溶液的涂层。在该实验中，一个ICAM-1/Fc的嵌合体溶液在25微克/毫升和P-Selectin/Fc嵌合体在PBS中0.5微克/毫升（pH8.0）中被使用。 </li><li>大衣与50微升底物的显着区域。孵育过夜，在4℃下烹调应在两天内被使用，并且涂覆区域不应该被允许干燥。如果要保持上盘的50微升的溶液加入PBS。 </li></ol> 4，中性粒细胞分离（在室温下进行所有步骤） <ol><li>获得知情同意后，收集参与者的血液通过静脉切开成Ñ​​抗凝血液收集管或真空采血管（EDTA或肝素）。采血后，分离前稀释血液1:1的PBS。 </li><li>准备一个两层聚蔗糖的50毫升离心管中分离外周血单个核细胞和中性粒细胞。首先添加15毫升沉重聚蔗糖（见材料清单，ρ= 1.118-1.120），然后仔细层10毫升光聚蔗糖（见材料清单，ρ= 1.077-1.080）在沉重的聚蔗糖的顶部。应的光聚蔗糖和沉重的Ficoll层之间的尖锐边界。最后，小心层25毫升稀释血样品上的光的Ficoll的顶部，而不会干扰的Ficoll层。 </li><li>离心管中，在250×g离心30分钟，在室温下进行。注：该离心机转子制动应该关闭这些自旋尽量减少在转子减速的细胞分离的离心年底的潜在干扰。离心后，将下面的层（从顶部到底部）存在：顶层是等离子体福卢由PBMC层上的光的Ficoll层的顶部周三，嗜中性粒细胞层用少量的红血细胞（RBC）的轻和重的Ficoll随后在管的底部重的Ficoll层和红细胞之间。 </li><li>收获和嗜中性粒细胞层转移到新的50ml管中有一个移液管，加PBS至50ml离心终体积在225×g下在室温下10分钟。 <ol><li>离心后，仍可能存在的RBC与嗜中性粒细胞混合。吸出上清液下至10ml刻度。通过管（或短暂低速涡旋）的温和搅拌悬浮的嗜中性粒细胞 - 红细胞沉淀，然后用50ml的PBS再次洗涤。 </li></ol></li><li>第二次洗涤后，吸出上清液。以除去污染的红细胞，通过搅拌（或短涡旋）重新悬浮沉淀的细胞中残留的PBS中，加入25毫升水2 O，轻轻涡旋10秒以裂解红细胞。 <ol><li>取25 mL 1.8％NaCl溶液中，并立即混匀通过离心分离，在225×g下在室温下10分钟。所述污染红细胞现在应该裂解留下白色中性粒细胞沉淀。 </li></ol></li><li>用PBS洗中性粒细胞的细胞沉淀。 </li><li>重悬分离的嗜中性粒细胞在RPMI培养基中含有10％FBS，并确定光学显微镜用血细胞计数下将细胞浓度。 </li><li>调整细胞密度至500,000个细胞/ ml用完全RPMI-10％FBS的培养基中。 </li></ol> 5，流室粘附实验<ol><li>装配流动室。将包含在显微镜上表中的融合内皮细胞或纯化的粘附受体的配体35毫米的菜。连接平行板流动腔与注射泵，真空系统，留下一行开的中性粒细胞输入。将流动室上盘的顶部，并拧紧流室组件13。 （ 见图1） </li><li>启动电脑连接吨的视频录制程序Ø显微镜。调整显微镜的领域和焦点，直到一个明确的字段具有完全生长的HUVEC细胞或一个字段内的配体涂层区域是可见的。 </li><li>使用注射泵，冲洗液流腔的RPMI培养基中。确保有室之内没有气泡或中性粒细胞输入线。 </li><li>如果需要的话，嗜中性粒细胞可引发的低剂量（10 -8 M）的fMLP 10分钟。这将允许为不同的供体14之间的中性粒细胞活化的基础水平的匹配。 </li><li>使用注射泵，以嗜中性粒细胞注入到流动室中限定的速度（350微升/分钟的速度，这等于1.5达因/厘米2的剪切应力是用在本研究中）。录制视频。因为中性粒细胞粘附可发生迅速，具有4-5分钟长度的视频通常是足够的定量粘附事件进行分析。 </li><li>贴壁细胞被定义为移动小于1细胞直径的小区5秒内对血管内皮细胞或配体涂层表面15,16。计数贴壁细胞的总数量在该领域目前在使用这个法则录制的视频。通过记录长度相似的影片，不同的捐助者的嗜中性粒细胞，就可以计算出贴壁细胞/分钟比较不同供体之间的粘合性。 </li></ol>

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作者什么都没有透露。

该协议将引导微创激活中性粒细胞的剪切应力条件下谨慎量化中性粒细胞粘附的分离和隔离。中性粒细胞粘附是在炎症的关键过程。因为在这一过程中的多个分子的遗传变异已经证明易患自身免疫病1,2的发展，一种能够定量地评估人类嗜中性粒细胞牢固粘附的测定系统是必需的。在本协议中所述的方法，允许细心和定量测定中性粒细胞在剪切应力下控制在体外环境中的公司胶粘剂潜力。这种方法从而允许基因型个体之间的定量中性粒细胞粘附的直接比较，以确定遗传变异的粘附分子14的重要性。 在这种方法的几个步骤，值得慎重考虑achievË高度量化和可重复性的结果。在血管内皮细胞的准备，关键是利用他们在流动室前达到100％的细胞汇合。对于使用涂有纯配体的表面，基材涂布面积不应该被允许干，以避免变性的配体。此外，人中性粒细胞的制备是将实验的成功至关重要。在从血液中分离嗜中性粒细胞的主要问题包括通过最小化的涡流，以避免活化，保持细胞在室温下（ 即血液不应该被储存在冰上和离心步骤应在室温下进行），并在完成隔离和实验轻轻处理在最短的时间成为可能。还有，也可用于制备细胞用于这些测定17,18附加的嗜中性粒细胞的分离方法。 使用新鲜分离的人中性粒细胞的实用角度出发，附着测定<html>应在3-6小时参与者放血后启动。嗜中性粒细胞是处理极为敏感，抽血和使用之间的时间延长可能会影响检测结果。前向流室法仔细测定中性粒细胞浓度的也是必要的，以实现准确和可重复的结果。 在流室法，仔细监视录像，以确保流动速率是一致的，有在整个实验的长度没有湍流是重要的。改变流速或湍流的存在下，将须，该实验重复进行。在实验后，这也很重要，以评估剩余的中性粒细胞在显微镜以确保该嗜中性粒细胞不结块。结块此时将表明，嗜中性粒细胞已成为激活从而在实验过程中可以显著改变嗜中性粒细胞的密度。 内容“&gt;流动室产生腔室中的接近均匀的剪切应力（τ=6Qμ/（WH 2），其中Q =流速，μ=动态粘度和流动室W =宽度，下h =高度流腔15），在我们的研究中，我们使用的350微升/分钟的中性粒细胞粘附的流速，从而产生1.5达因/厘米2的剪切应力（瓦特= 0.25厘米，H =0.01英寸，水的粘度在37℃（0.007泊）用作RMPI介质的粘度的近似值），对于一个特定的流室，可以改变流率，以实现不同程度的剪切应力，以模拟不同的生理条件下，典型的生理剪切应力在人类血管可之间0.5-5.0达因/厘米的范围13,16。剪应力在其他的血管和其他动物被列于表1 113,16,19 20。 而我们的方法集中于人类中性粒的附着性的研究LS，这个方法并不限于嗜中性粒细胞，并且可以很容易地应用于粘接或用简单的修改的其它细胞类型的轧制研究。此外，在该方法的基底可以为不同的目的而改变。 虽然这个协议是很容易适应不同的研究，有一定的局限性。因为在这里实现该协议要求大量的原代细胞进行分析。这可能会妨碍从小型动物的原代细胞的分析。此外，需要固定化的纯化的配体粘附在主动/可用的构象可能限制配体的范围。用Fc融合蛋白的大大增强了板表面上实现适当的配体构象的可能性。尽管如此，我们的方法具有显著的灵活性，允许的粘附事件的定量分析。这些研究将大大提高我们的理解粘附受体 - 配体对的，和遗传变异的潜在功能的重要性在这些蛋白质在人类疾病的发病机制。

在两个β2整合素和ICAM在配位体的遗传变异，现在认为与自身免疫性疾病1,2的发展相关联。在从与这些变体的个体来源的细胞这些变体的功能性后果的确定是必要的我们的这些变体如何有助于自身免疫性疾病的发病机制的理解。这种功能研究允许由自然发生的遗传变异塑造健康和疾病的免疫反应机制的决心。在SLE的具体例子，我们现在知道在ITGAM（细胞CD11b）的变种及其配体，ICAM-1，具有强烈的1,2疾病发展相关联。因为中性粒细胞是至关重要的炎症反应，中性粒细胞粘附的定量研究可提供机械洞察ITGAM / ICAM遗传变异如何改变炎症。 NeutropHIL牢固粘附于内皮细胞（EC）是一个高度管制的过程中起着炎症反应3,4至关重要的作用。中性粒细胞的粘附牢固如下初始中性粒细胞滚动和捕获欧共体，并最终可能导致轮回体内 。这些过程涉及许多不同种类的粘附分子，包括ICAM-1，ICAM-2，P-选择素，E-选择素在血管内皮细胞和β2整合素对嗜中性粒细胞5-9。因此，从捐助者与粘附分子的不同等位基因变异小心量化中性粒细胞粘附的将是重要的，了解这些遗传变异的功能和病理的后果。 实验使用一个流动室可以建立一种体外环境下用类似于血管体内环境10-12观察到的流体剪切应力。的确，一个流室法加上人类umbili卡尔静脉内皮细胞（HUVEC）可以模仿血管的体内环境。使用这种方法，可以研究对血管内皮细胞的总细胞的粘合性能。此外，该流动室的高度可控的环境也允许细胞进行评估结合到纯化的密合性配体，例如ICAM-1，以促进特异性受体 - 配体相互作用的研究。 我们在这里提出一个利用一个流室粘附实验系统，研究人末梢血嗜中性粒细胞的粘接性的HUVEC和纯化的配体基片的方法。使用这种方法与捐助者的细胞表达不同的粘附分子等位基因变异体使我们能够评估如何将这些基因变异可以改变人中性粒细胞粘附牢固。

<table border="0" cellpadding="0" cellspacing="0" style="width:677px;" width="677">
	<tbody>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:677px;">Table of Reagents Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Name of reagent</td>
			<td style="width:107px;">Company</td>
			<td style="width:111px;">Catalogue number</td>
			<td style="width:268px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">0.25% Trypsin/EDTA</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">25200-056</td>
			<td style="width:268px;">with Phenol Red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">2X Trypsin inhibitor</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">R-002-100</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">35mm tissue culture dish</td>
			<td style="width:107px;">Corning Inc.</td>
			<td style="width:111px;">430165</td>
			<td>Standard Tissue Culture Treated Surface</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:192px;">75cm2 tissue culture flask</td>
			<td style="width:107px;">Corning Inc.</td>
			<td style="width:111px;">430641</td>
			<td>Standard Tissue Culture Treated Surface</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">CCD camera</td>
			<td style="width:107px;">Dage-MTI</td>
			<td style="width:111px;">Model 300T-RC</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Cell freezing container&#160;</td>
			<td style="width:107px;">Biocision</td>
			<td style="width:111px;">BCS-045</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">EBM-2</td>
			<td style="width:107px;">Lonza Inc.</td>
			<td style="width:111px;">CC-3156</td>
			<td style="width:268px;">HUVEC growth basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">EGM-2 Bulletkit</td>
			<td style="width:107px;">Lonza Inc</td>
			<td style="width:111px;">CC-4176</td>
			<td style="width:268px;">HUVEC growth factors for basal medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">FBS</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">10082-147</td>
			<td style="width:268px;">Certified, Heat Inactivated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Gelatin</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">G9391</td>
			<td style="width:268px;">from bovine skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Hemacytometer</td>
			<td style="width:107px;">Fisher scientific</td>
			<td style="width:111px;">02-671-51B&#160;</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Fibronectin</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">F2006</td>
			<td style="width:268px;">from human plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Flow chamber</td>
			<td style="width:107px;">Glyco Tech</td>
			<td style="width:111px;">31-001</td>
			<td style="width:268px;">Circular flow chamber for 35mm dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">fMLP</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">47729</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Histopaque-11191</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">11191</td>
			<td style="width:268px;">Heavy ficoll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">HUVEC</td>
			<td style="width:107px;">Lonza Inc.</td>
			<td style="width:111px;">CC-2517A</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">ICAM-1</td>
			<td style="width:107px;">R&#38;D systems</td>
			<td style="width:111px;">720-IC</td>
			<td style="width:268px;">Fc chimera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Lymphocyte separation medium</td>
			<td style="width:107px;">Mediatech Inc.</td>
			<td style="width:111px;">25072CV</td>
			<td style="width:268px;">Light ficoll</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Microscope</td>
			<td style="width:107px;">Zeiss</td>
			<td style="width:111px;">Axiovert 100</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">RPMI 1640 medium</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">11875</td>
			<td style="width:268px;">with L-Glutamine and Phenol Red&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Protein A</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:111px;">P6031</td>
			<td style="width:268px;">resuspend in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">P-Selectin</td>
			<td style="width:107px;">R&#38;D systems</td>
			<td style="width:111px;">137-PS</td>
			<td style="width:268px;">Fc chimera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Syringe pump</td>
			<td style="width:107px;">KD Scientific</td>
			<td style="width:111px;">KDS270</td>
			<td style="width:268px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">TNF-a</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:111px;">PHC3015</td>
			<td style="width:268px;">Recombinant Human Protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:107px;">Life technologies</td>
			<td>15250-061</td>
			<td></td>
		</tr>
	</tbody>
</table>

human neutrophil flow chamber adhesion assay

嗜中性粒细胞牢固粘附到内皮细胞在炎症在健康和疾病的关键作用。中性粒细胞粘附牢固的过程中涉及到许多不同的粘附分子，包括β2整合素家族的成员和他们的反受体ICAM家庭。最近，在天然存在的遗传变异体β2整合素和ICAM的被报道与自身免疫性疾病相关联。因此，中性粒细胞从这些粘附分子的不同等位基因型个体的定量涂胶量是相对于自身免疫相关的发展机制研究的重要。在流动室系统粘附研究，可以创建具有相似于血管环境在体内观察到的流体剪切应力的环境。在这里，我们提出了一个方法，用流室法系统来研究人外周血中性粒细胞的定量粘接性能s到人脐静脉内皮细胞（HUVEC）和纯化的配体的底物。用这种方法，从供体与粘附受体的不同等位基因变体的中性粒细胞的粘合能力，可以评估和比较。这种方法也可以被修改，以评估其他主要类型的细胞或细胞系的附着。

嗜中性粒细胞的实例结合到纯化的配体（ICAM-1/P-Selectin）涂覆的流室（ 图2），嗜中性粒细胞结合到HUVEC涂覆流动腔实验（ 图3）或显示正在。如图所示，在图中，嗜中性粒细胞中不断积累/连续流动的条件下，附着在涂层表面或血管内皮细胞。根据我们的典型的实验条件下，我们可以观察到50-70人中性粒细胞牢固地附着在涂层表面或HUVEC在一个四分钟的记录时间。然而，嗜中性粒细胞粘附分子，或在衬底（纯化的配体或HUVEC）的等位变体的等位基因变体，可以显着地改变量化中性粒细胞粘附14。 我们还评估了时间在我们的研究中观察到的中性粒细胞粘附事件的依赖性。虽然我们维持恒定流量的条件下，它可能是细胞的粘合性能随时间而改变。 ħH但是，在我们的研究时间比较短的课程，我们没有观察粘连随时间的速度保持一致的任何差异。例如，评估黏附在1-2分钟的时间点相比，附着力3-4分钟的时间点之间观察到的是不是一致的不同。当然，如果粘附实验过程中被刺激的细胞，然后在一段时间内粘合性能的变化可以预期。 在我们的研究中，我们故意灌注细胞低剂量的fMLP（10 -8 M）为研究前10分钟控制了隔离引起的中性粒细胞活化。内皮细胞（HUVEC在我们的研究中）也需要事先激活上调黏附分子的表达以获得最佳白细胞牢固粘连。在没有预处理的，内皮细胞（HUVECs）将支持非常小的中性粒细胞粘附。在我们的研究中，我们使用10纳克/毫升TNFα的治疗在使用前4-6小时。 IL-1β（10毫微克/毫升）和<html>脂多糖（0.5-1微克/毫升），也可用于预激活的内皮细胞。重要的是，未经处理的HUVEC可作为阴性对照，以确保细胞的粘附是由特定的（受体介导的）的中性粒细胞 - 内皮细胞相互作用。可替代地，抗受体抗体可用于阻断特异性受体，以评估粘附特异性。 <img alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/51410/51410fig1highres.jpg" src="/files/ftp_upload/51410/51410fig1.jpg" /> 在显微镜舞台上图1。流室的配置。 <a href="https://www.jove.com/files/ftp_upload/51410/51410fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> oad/51410/51410fig2.jpg“/&gt; 0时间点，B：1分钟的时间点，C：2分钟的时间点和D。中性粒细胞粘附在不同时间点（A至ICAM-1/P-Selectin涂层表面的样本视频图2的屏幕快照 ： 4分钟的时间点）。 ICAM-1的浓度为25微克/毫升和P-选择素是0.5微克/毫升。中性粒细胞流速为350μL/分钟，为50万个/毫升中性粒细胞密度。 <img alt="图3" fo:content-width="6in" fo:src="/files/ftp_upload/51410/51410fig3highres.jpg" src="/files/ftp_upload/51410/51410fig3.jpg" /> 图3屏幕截图从嗜中性粒细胞粘附到血管内皮细胞表面的涂层在不同时间点（A：0时间点，B：1分钟的时间点，C：2分钟的时间点，和D：4分钟的时间点） 的一个样本视频 。中性粒细胞流速为350微升/毫升50万个细胞/ ml中性粒细胞密度。 <table border="0" cellpadding="0" cellspacing="0" ><tbody><tr ><td>种类</td><td >位置</td><td >剪切应力（达因/厘米2） </td></tr><tr ><td>人的</td><td>常见caroid动脉</td><td> 11.6 </td></tr><tr ><td>人的</td><td>鳃动脉</td><td> 6.5 </td></tr><tr ><td>人的</td><td>普通股动脉</td><td> 4.3 </td></tr><tr ><td>人的</td><td>肾上主动脉</td><td> 7.3 </td></tr><tr ><td>人的</td><td> Supraceliac主动脉</td><td> 4.2 </td></tr><tr ><td>人的</td><td>浅表fermoral动脉</td><td> 4.4 </td></tr><tr ><td>人的</td><td>静脉</td><td> 0.5-5.0 </td></tr><tr ><td>人的</td><td>视网膜动脉第一</td><td> 40.2 </td></tr><tr ><td>人的</td><td>视网膜动脉第二</td><td> 0.001 </td></tr><tr ><td>人的</td><td>视网膜静脉第一</td><td> 23.2 </td></tr><tr ><td>人的</td><td>视网膜静脉第二</td><td> 0.43 </td></tr><tr ><td>狗</td><td>常见caroid动脉</td><td> 15.8 </td></tr><tr ><td>兔子</td><td>常见caroid动脉</td><td> 23.3 </td></tr><tr ><td>鼠</td><td>常见caroid动脉</td><td> 46.​​6 </td></tr><tr ><td>鼠标</td><td>常见caroid动脉</td><td> 64.8 </td></tr><tr ><td>狗</td><td>普通股动脉</td><td> 9.8 </td></tr><tr ><td>兔子</td><td>普通股动脉</td><td> 156.8 </td></tr><tr ><td>鼠</td><td>普通股动脉</td><td> 65.9 </td></tr></tbody></table> 表1样品的剪切应力在不同器官和不同的物种。 *摘自参考文献13，16，19，和20。

Watch this Scientific Journal Video about Human Neutrophil Flow Chamber Adhesion Assay at JoVE.com

Human Neutrophil Flow Chamber Adhesion Assay

报道定量中性粒细胞粘附的方法。此方法创建类似于血管遇到了动态流环境。它使中性粒细胞粘附到任何纯化黏附分子（配体）或类似体内环境与剪切应力的上下文内皮细胞基板（HUVEC）的调查。

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 ...

human-neutrophil-flow-chamber-adhesion-assay

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  University of Alabama at Birmingham. 报道定量中性粒细胞粘附的方法。此方法创建类似于血管遇到了动态流环境。它使中性粒细胞粘附到任何纯化黏附分子（配体）或类似体内环境与剪切应力的上下文内皮细胞基板（HUVEC）的调查。 

视频: Human Neutrophil Flow Chamber Adhesion Assay

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

人类<em在体外承认免疫失衡的早期国家禁止作为筛检工具

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

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, ...

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 ...

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 ...

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 ...

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 ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

人外周血 NK 细胞活性评估流基于流式细胞仪检测细胞毒性检测

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

小鼠与人肺泡巨噬细胞的分离和体外培养

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

全骨髓的制备,用于中性粒系细胞的大规模细胞测定分析

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...