Name: quantifying human monocyte chemotaxis in vitro and murine lymphocyte trafficking in vivo
Uploaded: 2017-10-30T13:00:00.000+00:00
Duration: 8 min 38 s
Description: Watch this Scientific Journal Video about Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo at JoVE.com

这项工作得到了伯纳德. 施瓦茨计划的支持, 洛克菲勒大学的医生科学家, 洛克菲勒大学的罗伯逊治疗发展基金, 和克生物医学和营养学研究中心在洛克菲勒大学

所有程序均由洛克菲勒大学和 #39 的机构动物保育和使用委员会批准。所有动物都在特定的无病原体条件下居住. 
 1. 体外 趋化 <ol> <li> 区域性 THP-1 单元格 6 在罗斯威尔公园纪念学院 1640 (RPMI 1640) 补充10% 胎牛血清 (FBS) 和 4-(2-羟乙基)-1-piperazineethanesulfonic 酸 (HEPES; 25 毫米) (完整介质), 保持密度介于 0.5-1.0 x 10 6 单元格/mL 之间. </li>
	<li> 在35毫米细胞培养皿中, 标签 3.5 x 10 5 THP-1 细胞每条件与素乙酰 (素-AM; 2.5 和 #181; M) 或其他合适的荧光染料在完整的媒体37和 #176; C 为 30 min. 
	注意: 染料必须是细胞膜渗透性和与活细胞标签兼容. </li>
	<li> 预热一个平板阅读器到37和 #176; C. 
	注意: 除了温度控制之外, 还需要底层读取功能。请参阅步骤 1.10.2, 以了解用于量化迁移单元的平板阅读器的替代方法. </li>
	<li> 当细胞标记时, 准备化验板和下腔. 
	<ol> <li> 使用24个良好的组织培养板, 每个井都有一个条件。每三个条件执行一次. </li>
		<li> 添加750和 #181; 汉克和 #39 的缓冲盐溶液 (HBSS) 以25毫米 HEPES 和0.1% 牛血清白蛋白 (BSA) 缓冲到24井板。这担当基本的控制情况。
		<ol> <li> 将其他井用于比较器条件, 始终保持750和 #181 底部腔室的总体积; L. 
			注: 在这个例子中, 人类单核细胞趋化蛋白 protein-1 (MCP-1) 充当趋化 7 的阳性控制。
			<ol> <li> 通过将 HBSS 与 25 mM HEPES、BSA (0.1%) 和 MCP-1 (75 ng/毫升) 组合在一起来组装试剂混合物。使用成年牛血清 (2.7% v/v) 与 MCP-1 作为一个额外的阳性对照. 
				注意: 趋化因子和趋化因子浓度将取决于研究中的趋轴。冻干 MCP-1 是悬浮在蒸馏水与 BSA (0.1%), 使一个股票的解决方案, 最终浓度为50和 #956; 克/毫升。这可以稀释在水中的工作解决方案, 10 和 #956; 克/毫升。添加5.6 和 #956; 我的这个解决方案, 以较低的会议厅. </li>
			</ol> </li> </ol> </li> <li> 在底部室的组成完成后, 将一个多孔插入到每个井中, 确保插入的制表符与板的凹槽对齐. 
		注: 为插入物选择合适的孔径大小。对于单核细胞和淋巴细胞, 孔径大小为3和 #956; m 执行良好。一些细胞类型和/或趋化因子系统可能需要一个基体涂层表面的最佳迁移。例如, 为了测量 THP-1 单核细胞趋化向 MCP-1, 纤维连接蛋白或胶原涂层的刀片推荐。对于平板阅读器的定量, 插入必须有一个 light-impermeant 涂层, 如聚乙烯对苯二甲酸乙酯, 以防止检测的 non-migrating 细胞在上部商会 8 . </li>
	</ol> </li> <li> 在将单元格素为30分钟后, 将单元格悬挂转换为15mL 锥形管。离心细胞在 1000 x g, 2 分钟, 视觉检查细胞, 以确认摄取素 AM ( 图 1 ). </li>
	<li> 小心地用真空器去除上清液。在无血清 RPMI 1640 中, 用 HEPES 25 毫米补充标记细胞。离心细胞在 1000 x g 2 分钟, 并丢弃上清液. </li>
	<li> 重细胞在无血清 RPMI 1640 补充与 HEPES 25 毫米。计算单元格并调整音量, 使单元格的最终浓度为 1.0-1.5 x 10 6 单元格/mL. </li>
	<li> 免除250和 #956; L 细胞悬浮入插入物 (上部房间). </li>
	<li> 在所有上部腔都被填满后, 轻轻地抬起板, 检查底部是否存在可能干扰迁移和检测的气泡。要删除气泡, 首先尝试移除定位插入。如果不成功, 使用27克针刺穿气泡, 然后重新插入. </li>
	<li> 将板放入 5ml 37 和 #176; C 板读取器. 
	<ol> <li> 从底部获取荧光读数, 间隔为1-3 分钟。在使用素时, 分别将荧光激发和发射波长设置为 485 nm 和 520 nm。根据细胞类型和趋化因子系统, 趋性典型地发生 1-4 h ( 图 2A ). </li>
		<li> 作为使用平板读取器进行连续测量的替代方法, 使用倒置的荧光显微镜 ( 图 2B ) 对油井进行图像处理, 分别能够在 485 nm 和 520 nm 上激发和探测光。使用低放大倍数来捕获插入的底部的大部分。通过使用 ImageJ 或类似软件 9 处理图像来量化单元格. </li>
	</ol> </li> </ol> 2。小鼠淋巴细胞的过继转移 <ol> <li> 供体细胞的制备 <ol> <li> 麻醉8-12 周老的 C57BL/6J 雄性供体鼠用异氟醚麻醉 (1-3%) 使用汽化器。用脚趾捏确定适当的麻醉深度. </li>
		<li> 将鼠标置于仰卧位。用手术刀做中线切口, 并在左上腹间隙找到脾脏。把整个脾脏和地方全部切除 (RPMI 补充 10% FBS 和25毫米 HEPES; 1 毫升/脾脏) 在冰上。安乐通过斩首来麻醉供体动物. </li>
		<li> 将脾组织轻轻地通过40和 #181; m 尼龙网入完全媒介 (1 毫升/脾脏) 使用注射器柱塞的末端做一个单细胞悬浮并且转移到15毫升圆锥形管. </li>
		<li> 添加4卷氯化铵钾 (ACK) 裂解缓冲液, 并在室温下孵育5分钟以溶解红细胞. </li>
		<li> 在室温下离心 1000 x g 的细胞2分钟并丢弃上清液。如果红细胞留在颗粒, 重在 ACK 裂解缓冲液, 孵育5分钟的室温, 离心细胞在 1000 x g 2 分钟和丢弃上清. </li>
		<li> 将完整介质中的单元格重为 2-5x10 7 单元/mL, 然后通过40和 #181 将细胞悬浮变形; m 尼龙网格去除细胞碎片. </li>
		<li> 标签 1 x 10 7 与活细胞相容的荧光染料的细胞/受体小鼠, 在随后的固定时将保留, 如 5-chloromethylfluorescein 乙酸酯 (最终浓度2和 #956; M) 10 .孵育在37和 #176; C 为 30 min. 
		注意: 要跟踪多个细胞的数量, 使用其他的荧光染料与额外的细胞数量。例如, 部分供体细胞可以用 5-(和-6)-(((4-氯甲基) 苯甲酰胺) 甲基 (最终浓度2和 #956; M) 11 . </li>
		<li> 添加4卷 HBSS 补充25毫米 HEPES, 然后离心在 1000 x g 为 2 min 和放弃上清液。重颗粒在 HBSS 补充 HEPES 25 毫米到最后浓度 1 x 10 8 细胞/毫升。
		<ol> <li> 如果使用多个荧光染料, 请将单元格的数量保持分离, 直到注入之前. </li>
			<li> 将每个颜色的单元格的小分 (10 #956; L) 转换为固定液 (1 毫升), 用于以后的流式细胞术补偿. </li>
		</ol> </li> </ol> </li> <li> 转移淋巴细胞 <ol> <li> 麻醉8-12 周前的 C57BL/6J 雄性受体小鼠, 使用汽化器 (1-3%); 通过脚趾掐确定麻醉深度。将兽医软膏应用于动物和 #39 的眼睛, 防止干燥. </li>
		<li> 短暂的旋涡供体细胞 (1 s) 和转移到1毫升注射注射器与27克, 0.5 针, 结合差异标记细胞的人口, 如果适用。
		<ol> <li> 将混合单元中的小分 (10 #956; L) 转换为固定液 (1 毫升), 用于随后的流式细胞仪分析. </li>
		</ol> </li> <li> 将供体动物置于俯卧位置。将轻度的尾侧压力施加到眼部的皮肤背部, 使眼球微微凸出. </li>
		<li> 引导针内侧并注入100和 #181; 供体细胞悬浮液 L 轨道到每个接收鼠标。细胞也可以通过尾静脉注入. </li>
		<li> 逐渐撤回注射针, 并将鼠标单独放在回收笼中。监视动物直到它恢复知觉;一旦完全恢复, 将动物归还给社会住房. </li>
	</ol> </li> <li> 收集和分析 <ol> 后1小时, 麻醉接受异氟醚麻醉的小鼠 (1-3%) 使用汽化器; 用脚趾掐确定麻醉深度。收获脾脏 (或其他组织的利益) (步骤 2.1.2) 从每个接收鼠标和地方到完整的媒体 (1 毫升/收件人)。安乐通过斩首来麻醉动物. 
		注: 组织收获前较长的时间间隔将通过至少24小时增加组织积累. </li>
		<li> 将脾组织轻轻地通过40和 #181; m 尼龙网状成完整的媒体使用注射器柱塞的结束, 使一个单细胞悬浮和转移到15毫升锥形管. </li>
		<li> 添加4卷的 ACK 裂解缓冲液, 室温孵育5分钟。在室温下离心 1000 x g 的细胞悬浮液2分钟并丢弃上清. </li>
		<li> 重细胞在染色缓冲 (磷酸盐缓冲盐水补充与 1% FBS)。计算单元格并调整到最终浓度 3 x 10 7 单元/毫升。转移100和 #181; L 细胞悬浮到一个96井圆的底板. </li>
		<li> 用流式细胞仪分析所需标记 (参见材料表) 的染色单元。添加荧光共轭抗体 (见材料表) 与所需的标记和孵化在4和 #176; C 为20分钟在黑暗中。添加100和 #181; L 染色缓冲液, 离心 1000 x g 的细胞3分钟, 并丢弃上清. </li>
		<li> 重200和 #181 中的颗粒; L 染色缓冲液, 离心 1000 x g 的细胞3分钟, 并丢弃上清. </li>
		<li> 重125和 #181 中的颗粒; 使用标准程序使用流式细胞仪染色缓冲液和采集样品. 
		注: 绿色染料是兴奋的 488 nm 激光和发射检测与505和530/30 分色过滤器。橙色染料是兴奋的 561 nm 和排放是检测到的582/15 分色过滤器。
		<ol> <li> 使用从2.1.8.2 中保存的单元格来补偿每个荧光染料. 
			注: 使用 bead-based 补偿与荧光匹配的激励和发射光谱的标记染料将导致次优补偿. </li>
			<li> 使用来自步骤2.2.2.1 的已保存的输入单元格, 以准确确定在使用多个标记的填充 ( 图 3 ) 时差异标记的输入单元格的比率。根据标记为未标记的单元格的比率来计算组织特定的迁移 ( 图 4 ). </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Griffith, J. W., Sokol, C. L., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+and+chemokine+receptors:+positioning+cells+for+host+defense+and+immunity.">Chemokines and chemokine receptors: positioning cells for host defense and immunity.</a> Annu Rev Immunol. 32, 659-702 (2014).</li><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traffic+signals+for+lymphocyte+recirculation+and+leukocyte+emigration:+the+multistep+paradigm.">Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm.</a> Cell. 76 (2), 301-314 (1994).</li><li>Ponda, M. P., Breslow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+stimulation+of+CCR7+chemotaxis+due+to+coagulation+factor+XIIa-dependent+production+of+high-molecular-weight+kininogen+domain+5.">Serum stimulation of CCR7 chemotaxis due to coagulation factor XIIa-dependent production of high-molecular-weight kininogen domain 5.</a> Proc Natl Acad Sci U S A. 113 (45), E7059-E7068 (2016).</li><li>Boyden, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemotactic+effect+of+mixtures+of+antibody+and+antigen+on+polymorphonuclear+leucocytes.">The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.</a> J Exp Med. 115, 453-466 (1962).</li><li>Chen, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+chamber+assay.">Boyden chamber assay.</a> Methods Mol Biol. 294, 15-22 (2005).</li><li>Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>Leonard, E. J., Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+monocyte+chemoattractant+protein-1+(MCP-1).">Human monocyte chemoattractant protein-1 (MCP-1).</a> Immunol Today. 11 (3), 97-101 (1990).</li><li>Crouch, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+fluorescence+based+assay+of+neurite+formation.">An automated fluorescence based assay of neurite formation.</a> J Neurosci Methods. 104 (1), 87-91 (2000).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat Methods. 9 (7), 671-675 (2012).</li><li>Poot, M., Kavanagh, T. J., Kang, H. C., Haugland, R. P., Rabinovitch, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+cell+cycle-dependent+changes+in+cell+thiol+level+by+combining+a+new+laser+dye+with+Hoechst+33342.">Flow cytometric analysis of cell cycle-dependent changes in cell thiol level by combining a new laser dye with Hoechst 33342.</a> Cytometry. 12 (2), 184-187 (1991).</li><li>West, C. A., He, C., Su, M., Swanson, S. J., Mentzer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aldehyde+fixation+of+thiol-reactive+fluorescent+cytoplasmic+probes+for+tracking+cell+migration.">Aldehyde fixation of thiol-reactive fluorescent cytoplasmic probes for tracking cell migration.</a> J Histochem Cytochem. 49 (4), 511-518 (2001).</li><li>Kim, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+greater+chemotactic+network+for+lymphocyte+trafficking:+chemokines+and+beyond.">The greater chemotactic network for lymphocyte trafficking: chemokines and beyond.</a> Curr Opin Hematol. 12 (4), 298-304 (2005).</li></ol>

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免疫细胞迁移的定量化可以通过简单和快速的检测来实现, 这两种方法都是体外和体内。我们证明了体外对人单核细胞的趋化反应的 MCP-1 梯度和增强血清。在体内, 供体小鼠脾有差异标记和继过继转移后, 从接受动物中回收。
使用平板阅读器的优点是采样几个时间点 (像每年三十年代一样频繁) 和自动化迁移细胞的定量。这省却需要选择一个单一的时...

强健的免疫力需要复杂的时间和空间协调的各种细胞类型, 以适当的反应, 以伤害, 感染和产生发生。发现了几十种趋化因子受体及其相应的配体, 并提供了分子机制, 使特定的细胞能够在特定的时间内被定向到特定的组织中。因此, 研究趋化和迁移是免疫学研究的一个不可缺少的组成部分。事实上, 所描述的体外检测方法最近被用作筛选工具, 用于识别趋因子, 加速 T 细胞向 c-c 趋化因子19和 213的趋性。这里描述的方法的目的是允许定量评估免疫细胞趋化性在体外和在体内。
博伊登 (细胞迁移和侵入) 室检测是一种廉价, 可重现, 快速评估细胞迁移的方法4,5。在标准的化验中, 上部的腔室与细胞一起播种, 并由较低腔内的多孔插入物隔开, 细胞迁移到其中。在所需时间, 迁移到插入的底部的细胞可以固定和染色的光显微镜定量。但是, 此类测量将数据收集限制在单个端点, 这就排除了动态数据收集, 并且需要进行广泛的优化以确定最佳的分析时间点。在这里, 一些适应的描述, 允许 real-time, 定量和多路测量的趋化性在体外。
对于在体内的研究, 使用一个功能终点, 即特定组织室中细胞的具体积累。预标记的供体细胞通过过继转移引入受体动物。这些供体细胞随后可以通过流式细胞术在受体组织收获后进行识别。还提出了一个 co-labeling 的战略, 允许确定在单一受体动物的不同细胞类型的贩运。这种方法消除了细胞注射的 inter-animal 变异, 并解释了生理 inter-animal 变异性。

<table><tbody><tr><td>RPMI-1640</td><td>Thermo Fisher Scientific</td><td>11875-093</td><td></td></tr><tr><td>HEPES</td><td>Thermo Fisher Scientific</td><td>15630-080</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>ATCC</td><td>30-2020</td><td></td></tr><tr><td>Cell culture flask</td><td>Corning</td><td>353136</td><td></td></tr><tr><td>Calcein AM</td><td>Thermo Fisher Scientific</td><td>C1430</td><td>Excitation: 485nm, Emission: 520nm</td></tr><tr><td>Cell culture dish</td><td>Corning</td><td>1007</td><td></td></tr><tr><td>24 well plate</td><td>Corning</td><td>353504</td><td></td></tr><tr><td>Fluoroblok Fibronectin Insert</td><td>Corning</td><td>CB354597</td><td></td></tr><tr><td>15 mL conical tube</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Cell Signaling Technology</td><td>9998S</td><td></td></tr><tr><td>Human Recombinant MCP-1</td><td>Peprotech</td><td>300-04</td><td></td></tr><tr><td>Adult bovine Serum</td><td>Sigma</td><td>B9433</td><td></td></tr><tr><td>HBSS with calcium and magnesium; no phenol red</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>SpectraMax M2e plate reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Olympus IX71 inverted fluorescence microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Isothesia</td><td>Henry Schein animal health</td><td>11695-6776-2</td><td>Isofluorane anesthesia</td></tr><tr><td>Cell strainer 40um nylon</td><td>Falcon Corning</td><td>352340</td><td></td></tr><tr><td>ACK lysing buffer</td><td>Quality Biological</td><td>118-156-101</td><td></td></tr><tr><td>CellTracker Orange CMTMR Dye</td><td>Thermo Fisher Scientific</td><td>C2927</td><td>Excitation: 541nm, Emission: 565nm</td></tr><tr><td>CellTracker Green CMFDA Dye</td><td>Thermo Fisher Scientific</td><td>C2925</td><td>Excitation: 485nm, Emission: 520nm</td></tr><tr><td>5ml syringe</td><td>BD Syringe</td><td>309646</td><td></td></tr><tr><td>1ml TB syringe</td><td>BD Syringe</td><td>309625</td><td></td></tr><tr><td>THP-1 cell line</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Brilliant Violet 421 anti-mouse CD3 Antibody</td><td>Biolegend</td><td>100228</td><td>Excitation: 405nm, Emission: 421nm</td></tr><tr><td>Brilliant Violet 605 anti-mouse CD19 Antibody</td><td>Biolegend</td><td>115540</td><td>Excitation: 405nm, Emission: 603nm</td></tr><tr><td>96-well round bottom plate</td><td>Corning</td><td>353077</td><td></td></tr><tr><td>LSRII</td><td>BD Biosciences</td><td></td><td>Flow cytometer</td></tr></tbody></table>

quantifying human monocyte chemotaxis in vitro and murine lymphocyte trafficking in vivo

趋化是沿着特定的化学渐变1进行迁移。趋化因子是趋细胞素, 促进细胞贩运的解剖和时间特异性2。趋化性是淋巴细胞和其他免疫细胞的重要功能, 可以定量评估体外。这份手稿描述的方法, 允许评价的趋化性, 无论是体外和在体内, 不同的细胞类型, 包括细胞株和本机细胞。体外, plate - based 格式允许在 real - time 中同时进行多个条件的比较 , 并且可以在 1 - 4 小时内完成 .体外化验条件可以纵引入激动剂和拮抗药 , 以及区分趋化与 chemokinesis, 这是随机运动。对于体内的贩运评估, 免疫细胞可以用多种荧光染料标记, 并用于过继转移。细胞的差别标记允许混合细胞的人口被引入到相同的动物, 从而减少方差和减少动物的数量, 需要一个充分的动力实验。迁移到淋巴组织发生在1小时, 和多个组织隔间可以取样。组织收获后的流式细胞术可以快速定量分析多种细胞类型的迁移模式。

当使用素染料时, 细胞的视觉检查将确认标签的摄取 (图 1)。自动荧光读数将跟踪迁移, 随着时间的推移, 随着细胞的底部插入。这些数据清楚地显示了诱导细胞迁移向 MCP-1, 以及增加这种反应的血清 (图 2A)。根据迁徙刺激的强度, 在信号增加之前至少有15分钟的滞后。荧光信号可能会峰值, 然后逐渐下降。这代表了附着在插入物底...

Watch this Scientific Journal Video about Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo at JoVE.com

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

淋巴细胞趋化和迁移定量评估的协议是免疫学研究的重要工具。在这里, 一个体外协议的描述, 允许 real-time, 多路复用的细胞迁移评估, 以及一个互补的体内技术, 使本机细胞跟踪脾脏。

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

quantifying-human-monocyte-chemotaxis-vitro-murine-lymphocyte

Research

JoVE Journal

Immunology and Infection

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

7.1K 次观看.  The Rockefeller University. 淋巴细胞趋化和迁移定量评估的协议是免疫学研究的重要工具。在这里, 一个体外协议的描述, 允许 real-time, 多路复用的细胞迁移评估, 以及一个互补的体内技术, 使本机细胞跟踪脾脏。

视频: Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

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

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

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

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

迁移人T淋巴细胞分离，培养和分析<em在体外</em

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

科研

免疫与感染

Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Here, we present an integrated protocol that measures monocyte subpopulation trafficking under flow in vitro by use of specific surface markers and confocal fluorescence microscopy. This protocol can be used to explore sequential recruitment steps as well as to profile other leukocyte subtypes using other specific surface markers.

体外流动下单核细胞亚群的同时研究

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

小鼠巨噬体化学术的延时成像

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

医学

阐明 T 细胞动力学:实时迁移分析方法

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

作者聚焦:通过实时分析 T 细胞迁移了解疾病机制

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

原代小鼠 CD8+ T 细胞的实时体外迁移测定

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

摘要

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此视频中的章节

迁移人T淋巴细胞分离，培养和分析

慢性炎症状态个体的单核细胞轮回和泡沫形成的定量研究

以层流为基础的检测培养血管细胞和粘附血小板白细胞的研究

体外流动下单核细胞亚群的同时研究

单核细胞化学策略活性在Vivo的定量和血液单核细胞衍生巨噬细胞的特征

外迁移系统中淋巴细胞迁移的评估

小鼠巨噬体化学术的延时成像

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

原代小鼠 CD8+ T 细胞的实时体外迁移测定

原代小鼠 CD8+ T 细胞的实时体外迁移测定