我们感谢Kim实验室的前任和现任成员，他们随着时间的推移为这些协议的开发做出了贡献。代表性数据由P01 AI102851/AI/NIAID NIH HHS/United States和P01 HL018208/HL/NHLBI NIH HHS/United States提供。该出版物的部分原因在于机构 Ruth L. Kirschstein 国家研究服务奖的资助编号 T32 GM135134。

动物协议得到了罗切斯特大学动物资源委员会的批准。本研究中的小鼠保持在罗切斯特大学动物设施的无病原体空间中。本研究使用雄性/雌性 C57BL/6 小鼠，年龄为 6-12 周 （15-30 g）。小鼠组织分离可以在工作台上进行，手套覆盖双手，面罩覆盖鼻子和嘴巴，或在生物安全柜内进行。所有细胞培养和板制备必须在生物安全柜中进行。本研究中使用的试剂和设备列在 材料表中。
1. CD8+ T细胞纯化和活化
<ol><li>材料准备<ol><li>对于CD8 + T细胞的阴性选择：从杂交瘤细胞系GK1.5和M5/114抗体的上清液中制备阴性选择培养基，它们分别产生抗CD4和抗MHCII。以1：1的比例混合上清液，过滤，并将其储存在4°C以备将来使用（>0.5μg每种抗体/百万总细胞）。或者，也可提供商业 CD8+ T 细胞分离试剂盒。</li>
		<li>对于T细胞的活化：将500μL的CD3（10μg/ mL）和CD28（16μg/ mL）抗体在1x DPBS（不含钙和镁）中添加到非组织培养（TC）处理的24孔板中，并在4°C下储存过夜以确保板结合。 注：抗体和蛋白质比常规 TC 处理的培养板/培养皿更好地包被在未经 TC 处理的培养板/培养皿上，因此未经 TC 处理的培养板用于 T 细胞生成。</li>
	</ol></li>
	<li>通过将 70 μm 细胞过滤器放入培养皿中来制备 10 cm 培养皿（或等效物），并将 2 mL R9 培养基分配到过滤器中（R9 培养基：RPMI 1640x 补充有 10% FBS、1% 抗生素 - 抗真菌剂、20 mM HEPES 缓冲液、1% MEM 非必需氨基酸、50 μM β-巯基乙醇）。</li>
	<li>获得由实验设计确定的具有适当遗传背景、性别、年龄和体重的小鼠。</li>
	<li>使用 CO2 对小鼠实施安乐死，然后进行颈椎脱位，或根据当地动物护理和使用协议定义并经该机构批准的适当安乐死策略。</li>
	<li>将鼠标置于仰卧姿势，将所有四个肢体垂直于身体伸展，并使用鼠标解剖 T 销或等效物将脚垫固定在解剖板上。用小鼠外科手术解剖剪刀在腹侧下腹部的皮肤层做一个浅表的中央切口，并直接切到下巴（~8 cm）。将皮肤与腹膜内膜分开，露出淋巴结。将皮肤垂直拉伸到远离躯干的地方，然后将其固定5.<ol><li>使用钝钳（或标准小鼠手术解剖套件中的首选类型）轻轻切除颈部、轴向、臂部和腹股沟淋巴结。转移到步骤 1.2 中准备好的细胞过滤器中。</li>
		<li>打开腹膜，切除脾脏。转移到步骤 1.2 中准备好的细胞过滤器中。</li>
		<li>将小鼠尸体丢弃在适当的生物危害容器中。</li>
	</ol></li>
	<li>通过在70μm细胞过滤器上用步骤1.2的2mL R9培养基机械破碎来制备次级淋巴组织的单细胞悬浮液，方法是将组织破碎工具顺时针和逆时针旋转半圈。 注意：鲁尔锁注射器的柱塞端，3 mL 或 10 mL，可以很好地粉碎和均质化组织。用户可以自行决定使用替代材料。</li>
	<li>用 7 mL 培养基清洗细胞、注射器端、过滤器和培养皿，确保过滤器保持直立，以防止可能存在的较大组织块或细胞聚集体转移到单细胞悬液中。</li>
	<li>将细胞悬液转移到 15 mL 锥形管中。</li>
	<li>在室温（20°C）下以270× g沉淀细胞5分钟，并倒出上清液。</li>
	<li>加入 500 μL 裂解缓冲液 1 分钟以去除红细胞。用 9.5 mL R9 培养基稀释，并以 270 x g （20 °C） 离心 5 分钟。倒出上清液。</li>
	<li>将沉淀重悬于10mL先前制备的含有抗MHCII和抗CD4的阴性选择培养基中（步骤1.1.1）。在室温（20°C）下岩石30分钟。</li>
	<li>将细胞以 270 x g 沉淀 5 分钟并倒出上清液。用 10 mL R9 培养基洗涤 3 次。</li>
	<li>同时，在 15 mL 锥形管中，在 7 mL 培养基中洗涤 200 μL 绵羊抗大鼠 IgG 珠。将管子放在磁铁上，取出介质，然后重复洗涤步骤两次。将珠子重悬于 7 mL R9 培养基中。</li>
	<li>将细胞在270× g（20°C）下沉淀5分钟，倒出上清液，然后将沉淀重悬于步骤1.13的7mL珠状悬浮液中。在室温（20°C）下岩石45分钟。</li>
	<li>通过磁珠耗尽富集 CD8+ T 细胞。将锥形管直接放在磁铁上，不带盖 3 分钟，以去除抗体/珠子结合的细胞。应保留 CD8+ 细胞。将试管留在磁铁中，使用血清移液管或等效物收集细胞悬液，并将其转移到新的 15 mL 试管中。以270× g（20°C）离心5分钟，并在培养基中洗涤3次。</li>
	<li>用1x DPBS清洗预涂层活化板（步骤1.1.2）两次，而不直接在板的底面移液。</li>
	<li>可选：执行活/死淋巴细胞分离以去除死细胞。<ol><li>将细胞转移到 15 mL 锥形管中。轻轻地将 2 mL 淋巴细胞分离培养基加入细胞悬液的底部。在低加速和减速下以900× g 离心10分钟（在室温下）。 注意：活细胞将在培养基和分离培养基的界面（中间白色层）处分离。死细胞会在试管底部沉淀，最后可能会被丢弃。</li>
		<li>将含有活淋巴细胞的中间层转移到新管中，并在室温下以270× g 离心5分钟。倒出上清液。</li>
	</ol></li>
	<li>将沉淀重悬于含有 10 U/mL 重组小鼠 IL-2 的 12 mL 培养基中。在未经 TC 处理的 24 孔板中每孔板 1 mL，并转移至 37 °C 过夜。</li>
</ol>2. 提升活化的 CD8+ T 细胞
<ol><li>使用标准台式光学显微镜，使用 4 倍或 10 倍物镜观察细胞。 注意：与灭活的 T 细胞相比，活化的 T 细胞看起来更大、更圆或更细长，并且在整个孔中应该有密集的细胞簇。</li>
	<li>要取出活化细胞，请用 1 mL 移液管搅碎细胞，确保在板边缘充分混合。将细胞转移到新的 15 mL 锥形管中。 注意：活化的细胞更粘，需要更剧烈的移液。</li>
	<li>用 1 mL R9 培养基洗涤孔以去除任何残留的细胞。如果需要，请重复一次。将细胞以270× g （20°C）离心5分钟。丢弃上清液。</li>
	<li>执行活/死淋巴细胞分离以去除死细胞，如步骤1.17-1.18所示。</li>
	<li>在非 TC 6 孔板中，在含有 IL-2 的 R9 培养基中每 3-4 天通过活/死细胞去除来维持 T 细胞。 注意：在早期激活期间，T 细胞在小尺寸孔中增殖得更好，可能是由于 T 细胞-T 细胞接触或 T 细胞衍生的可溶因子有助于它们的激活。快速 T 细胞增殖导致培养基在培养后 24-48 小时内变黄。此时，提起细胞并将其转移到具有足够新鲜培养基（通常为每孔 5 mL）的较大孔（未包被、非 TC 6 孔板）中以补料细胞。每当培养基出现黄色颜色变化时，或每 3-4 天更换一次新鲜培养基。</li>
</ol>3.玻璃皿的准备
<ol><li>通过在4°C下用300μL /cm 2 蛋白A（0.1mg / mL）包被过夜来制备玻璃细胞迁移室或板，通过分配到培养皿上并用1x DPBS洗涤两次并倾析，然后再进入下一步。</li>
	<li>用300μL /cm 2 重组小鼠ICAM-1或VCAM-1 Fc嵌合体（0.1-2.75mg / mL）与重组小鼠趋化因子（0.1-10μg/ mL）一起包被迁移室或板2-3小时在室温下。将其他整合素配体如纤连蛋白，小鼠胶原IV和小鼠E-钙粘蛋白制备，以在4°C下以2.5-10μg/ mL直接包被腔室过夜。<ol><li>用 1x DPBS 洗涤室两次，方法是分配到培养皿上并在加入细胞之前立即倾析。 注：根据激活、分化、致敏和启动的状态，T 细胞在不同浓度的整合素配体和趋化因子中可以以不同的方式迁移。强烈建议在多种不同浓度下测定细胞迁移。</li>
	</ol></li>
	<li>如果将T细胞与其他细胞（如癌细胞）共培养，则在成像开始前24小时将贴壁细胞铺在玻璃涂层板上，以实时方式测量T细胞对癌细胞的杀伤。</li>
</ol>4. 细胞的制备
<ol><li>可选：在37°C下，用每1 x 107 个细胞1μg/ mL的细胞追踪染料在1x DPBS中对T细胞和贴壁细胞进行染色15分钟，或根据制造商的方案。</li>
	<li>使用血细胞计数器或等效物对细胞进行计数。</li>
	<li>在37°C室中将1 x 105 个CD8 + T细胞（或所需量）铺在补充有1-5g / L葡萄糖的Leibovitz's L-15培养基中。 注：L-15 培养基配制为不含 CO2-碳酸氢钠系统。它使用游离碱性氨基酸、磷酸盐缓冲液和半乳糖进行缓冲，以维持 pH 值。</li>
	<li>可选：执行整合素阻断。<ol><li>用针对整合素的阻断抗体（1-10μg/ mL，抗小鼠CD11a（M17 / 4），抗小鼠CD18（M18 / 2），抗小鼠VLA-4（9C10）预孵育T细胞10分钟，并允许在同一抗体存在下爬行。</li>
	</ol></li>
</ol>5. 延时显微镜
<ol><li>进行视频显微镜检查。</li>
	<li>对于 T 细胞，每 10-60 秒采集一次明场和荧光图像，持续 10-60 分钟，或所需的采集设置。 注：T细胞迁移对温度敏感，小鼠T细胞迁移的最佳温度为37°C。 该协议使用的温度控制系统由一个封闭和绝缘的显微镜甲板和一个附带的加热系统组成。成像室位于成像台上。该腔室的周边有一个装满蒸馏水的井，在成像过程中允许一致的热量和湿度。微孔位于成像室的中心。</li>
</ol>6. 软件辅助分析T细胞迁移
<ol><li>使用Volocity软件评估T细胞迁移。<ol><li>打开 Volocity 并创建一个 新的图像序列。</li>
		<li>选择 所有延时视频显微镜视频并将其 移动到 Volocity的蓝色区域。</li>
		<li>使用对比度增强工具根据研究人员所需的设置调整亮度和对比度。</li>
		<li>检查图像尺寸：20 倍为 0.325 μm，10 倍为 0.65 μm。</li>
		<li>序列：设置时间点（例如：如果每 15 秒拍摄一张图像，持续 20 分钟，则为 81 张图像，每分钟 4 张）。</li>
		<li>取消选中测量选项卡中的所有时间点。</li>
		<li>使用强度找到对象 - 滑动条形图，使其刚好位于峰值。</li>
		<li>按大小排除物体：直径小于 10 μm 且大于 100 μm 的所有细胞，以排除细胞碎片或非细胞颗粒。排除迁移时间少于记录时间 20% 的 T 细胞（这可能因每个研究者的定义而异）。</li>
		<li>忽略静态对象 （复选框）。</li>
		<li>自动连接断开的区域 （复选框）。</li>
		<li>将最短路径设置为不超过 20 μm。</li>
		<li>细胞迁移的轨迹是连接同一细胞位置随时间推移的线。确保跟踪算法通过检测每个帧中通过分割检测到单个对象的位置进行跟踪，并且对象在帧之间匹配。</li>
		<li>通过选择“测量值”>“保存协议”>“命名新协议”来保存新协议。</li>
		<li>单击“测量”选项卡中的“测量所有时间点”。</li>
		<li>按时间跨度（从高到低）对轨道进行排序。</li>
		<li>记录研究者定义的具有良好轨迹的所有单元格的轨迹 ID 号。在自动小区跟踪之后，需要对每个小区轨迹进行手动评估，以排除错误识别或损坏的轨迹。排除具有不良轨道的单元格。</li>
		<li>将文件导出为逗号分隔的文本，并将数据传输到所需的分析软件。 注： 基本参数：速度 （μm/min）、位移（净移动，μm）、轨迹长度（总路径长度，μm）和蜿蜒指数（MI; 净位移/轨迹长度。MI = 1 表示完全直线轨道）。</li>
	</ol></li>
	<li>使用图像 J 执行手动跟踪。<ol><li>打开插件并选择 跟踪 和 手动跟踪。设置参数 - 时间间隔、x/y 校准（像素大小）和 z 校准（在手动跟踪模式下）。通过单击 “添加轨道”开始单个单元格跟踪，在第一个时间点选择一个单元格，然后在所有时间点继续进行。接下来，单击 “结束轨道”。对所有感兴趣的单元格重复此操作。 注意：Trackmate 是另一个用于细胞跟踪的工具，是一种 通过 ImageJ 插件提供的自动跟踪软件。</li>
	</ol></li>
</ol>

阐明 T 细胞动力学：实时迁移分析方法

<ol>
	<li>Sun, L., Su, Y., Jiao, A., Wang, X., Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+in+health+and+disease.">T cells in health and disease.</a> Signal Transduct Target Ther. 8 (1), 235 (2023).</li><li>Fowell, D. J., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatio-temporal+control+of+effector+T+cell+migration.">The spatio-temporal control of effector T cell migration.</a> Nat Rev Immunol. 21 (9), 582-596 (2021).</li><li>Tabdanov, E. D., 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=Engineering+t+cells+to+enhance+3d+migration+through+structurally+and+mechanically+complex+tumor+microenvironments.">Engineering t cells to enhance 3d migration through structurally and mechanically complex tumor microenvironments.</a> Nat Commun. 12 (1), 2815 (2021).</li><li>Kameritsch, P., Renkawitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+leukocyte+migration+strategies.">Principles of leukocyte migration strategies.</a> Trends Cell Biol. 30 (10), 818-832 (2020).</li><li>Flaherty, S., Reynolds, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+naive+CD4++T+cell+isolation+and+in+vitro+differentiation+into+t+cell+subsets.">Mouse naive CD4+ T cell isolation and in vitro differentiation into t cell subsets.</a> J Vis Exp. (98), e52739 (2015).</li><li>Sailer, C. 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=Pd-1(hi)+CAR-T+cells+provide+superior+protection+against+solid+tumors.">Pd-1(hi) CAR-T cells provide superior protection against solid tumors.</a> Front Immunol. 14, 1187850 (2023).</li><li>Lim, K., Hyun, Y. M., Lambert-Emo, K., Topham, D. J., Kim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+integrin+mac-1+in+vivo.">Visualization of integrin mac-1 in vivo.</a> J Immunol Methods. 426, 120-127 (2015).</li><li>Lim, K., 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=Neutrophil+trails+guide+influenza-specific+CD8++T+cells+in+the+airways.">Neutrophil trails guide influenza-specific CD8+ T cells in the airways.</a> Science. 349, 4352 (2015).</li><li>Lim, K., 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=In+situ+neutrophil+efferocytosis+shapes+T+cell+immunity+to+influenza+infection.">In situ neutrophil efferocytosis shapes T cell immunity to influenza infection.</a> Nat Immunol. 21 (9), 1046-1057 (2020).</li><li>Reilly, E. C., 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=T(rm)+integrins+CD103+and+CD49a+differentially+support+adherence+and+motility+after+resolution+of+influenza+virus+infection.">T(rm) integrins CD103 and CD49a differentially support adherence and motility after resolution of influenza virus infection.</a> Proc Natl Acad Sci U S A. 117 (22), 12306-12314 (2020).</li><li>Amitrano, A. M., 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=Optical+control+of+CD8(+)+T+cell+metabolism+and+effector+functions.">Optical control of CD8(+) T cell metabolism and effector functions.</a> Front Immunol. 12, 666231 (2021).</li><li>Xu, 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=Optogenetic+control+of+chemokine+receptor+signal+and+t-cell+migration.">Optogenetic control of chemokine receptor signal and t-cell migration.</a> Proc Natl Acad Sci U S A. 111 (17), 6371-6376 (2014).</li><li>Capece, T., 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+novel+intracellular+pool+of+IFA-1+is+critical+for+asymmetric+CD8(+)+T+cell+activation+and+differentiation.">A novel intracellular pool of IFA-1 is critical for asymmetric CD8(+) T cell activation and differentiation.</a> J Cell Biol. 216 (11), 3817-3829 (2017).</li><li>Hirai, T., Whitley, S. K., Kaplan, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+function+of+memory+CD8(+)+T+cells+in+skin.">Migration and function of memory CD8(+) T cells in skin.</a> J Invest Dermatol. 140 (4), 748-755 (2020).</li><li>Mouchacca, P., Schmitt-Verhulst, A. M., Boyer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cytolytic+T+cell+differentiation+and+granule+exocytosis+with+T+cells+from+mice+expressing+active+fluorescent+granzyme+B.">Visualization of cytolytic T cell differentiation and granule exocytosis with T cells from mice expressing active fluorescent granzyme B.</a> PLoS One. 8 (6), e67239 (2013).</li><li>Nuzzi, P. A., Lokuta, M. A., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+neutrophil+chemotaxis.">Analysis of neutrophil chemotaxis.</a> Methods Mol Biol. 370, 23-36 (2007).</li><li>Heit, B., Tavener, S., Raharjo, E., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intracellular+signaling+hierarchy+determines+direction+of+migration+in+opposing+chemotactic+gradients.">An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients.</a> J Cell Biol. 159 (1), 91-102 (2002).</li><li>Zigmond, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+polymorphonuclear+leukocytes+to+orient+in+gradients+of+chemotactic+factors.">Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors.</a> J Cell Biol. 75 (2), 606-616 (1977).</li><li>Jowhar, D., Wright, G., Samson, P. C., Wikswo, J. P., Janetopoulos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+access+microfluidic+device+for+the+study+of+cell+migration+during+chemotaxis.">Open access microfluidic device for the study of cell migration during chemotaxis.</a> Integr Biol (Camb). 2 (11-12), 648-658 (2010).</li><li>Sai, J., Walker, G., Wikswo, J., Richmond, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+IL+sequence+in+the+LLKIL+motif+in+CXCR2+is+required+for+full+ligand-induced+activation+of+Erk,+Akt,+and+chemotaxis+in+HL60+cells.">The IL sequence in the LLKIL motif in CXCR2 is required for full ligand-induced activation of Erk, Akt, and chemotaxis in HL60 cells.</a> J Biol Chem. 281 (47), 35931-35941 (2006).</li><li>Choi, Y., Sunkara, V., Lee, Y., Cho, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exhausted+mature+dendritic+cells+exhibit+a+slower+and+less+persistent+random+motility+but+retain+chemotaxis+against+ccl19.">Exhausted mature dendritic cells exhibit a slower and less persistent random motility but retain chemotaxis against ccl19.</a> Lab Chip. 22 (2), 377-386 (2022).</li><li>Harding, M. G., Zhang, K., Conly, J., Kubes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+crawling+in+capillaries:+A+novel+immune+response+to+staphylococcus+aureus.">Neutrophil crawling in capillaries: A novel immune response to staphylococcus aureus.</a> PLoS Pathog. 10 (10), e1004379 (2014).</li><li>Rommerswinkel, N., Niggemann, B., Keil, S., Zanker, K. S., Dittmar, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration+within+a+three-dimensional+collagen+matrix.">Analysis of cell migration within a three-dimensional collagen matrix.</a> J Vis Exp. (92), e51963 (2014).</li><li>Wu, P. H., Giri, A., Sun, S. X., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+cell+migration+does+not+follow+a+random+walk.">Three-dimensional cell migration does not follow a random walk.</a> Proc Natl Acad Sci U S A. 111 (11), 3949-3954 (2014).</li><li>Lammermann, T., 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Overstreet, M. 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=Inflammation-induced+interstitial+migration+of+effector+CD4(+)+T+cells+is+dependent+on+integrin+&#945;v.">Inflammation-induced interstitial migration of effector CD4(+) T cells is dependent on integrin &#945;v.</a> Nat Immunol. 14 (9), 949-958 (2013).</li></ol>

作者声明，所有研究和手稿准备都是在没有任何可能被解释为潜在利益冲突的商业或财务关系的情况下进行的。

理解体内收敛信号的生物学影响具有挑战性，而且不容易解释。本文介绍的方案提供了一种合理的方法来理解在高度定义和生物学相关条件下的 T 细胞迁移。这些条件可以根据研究者的判断来指定，并且可以修改方案以适应各种 T 细胞群、激活状态和细胞表型的需求。此外，许多配体和受体可以通过这些定义的抗体阻断 7,8 和配体包被玻璃程序<su...

T细胞是适应性抗原特异性免疫反应的主要效应因子。在群体水平上，T细胞是异质的，由具有不同特化功能的细胞亚群组成。重要的是，CD8+ T 细胞是免疫系统的主要细胞溶解效应因子，可直接消除感染或功能失调的细胞1。
成熟的CD8+ T细胞存在于组织中，并通过血液和淋巴管循环以寻找抗原。在感染过程中，T 细胞在血液或组织中呈递抗原，并迅速流向脾脏或最近的引流淋巴结，开始产生有效的免疫反应。在任何一种情况下，T细胞都会被激活，进行克隆扩增，并离开淋巴系统进入血液，如果还没有的话。在此过程中，细胞内信号传导导致淋巴归巢受体下调，并上调许多对组织特异性迁移至关重要的整合素和趋化因子受体2。最终，T 细胞向感染部位的定向迁移是由包括整合素和趋化因子信号传导在内的收敛环境信号驱动的。
趋化因子大致可分为两大类：（1） 稳态信号，对分化、存活和基础功能至关重要，以及 （2） 炎症信号，如 CXCL9、CXCL10 和 CCL3，是趋化性所必需的。通常，趋化因子除了激活整合素表达外，还会产生驱动定向迁移的信号梯度，称为趋化性1。趋化性受到精细调节且高度敏感，T细胞能够对梯度的微小变化做出反应，这些变化可以引导它们朝向特定方向或位置。
除了这些 T 细胞相关因素外，迁移还受到细胞外基质 （ECM） 组成和密度的影响。ECM 由密集的蛋白质网络组成，包括胶原蛋白和蛋白聚糖，它们为 T 细胞上的粘附整合素受体提供支架。整合素是一个多样化的跨膜蛋白家族，每个跨膜蛋白都具有高度特化的结合域和下游信号转导效应。整合素受体在 T 细胞表面的动态表达使其能够快速适应其不断变化的环境3.重要的是，整合素将 ECM 和细胞内细胞骨架肌动蛋白网络连接起来，它们共同产生 T 细胞运动所需的推进力。
总之，迁移模式根据免疫细胞表型或环境信号而变化。这些复杂的生物过程受到 T 细胞、周围细胞和局部感染组织表面细胞因子、趋化因子和整合素表达的严格调控。 在体内，这些迁移机制可能很复杂，并且可能由几个加性信号4 产生。由于这种复杂性，不可能在看似环环相扣的变量之间建立因果关系。为了克服这个问题，有几种 体外 方法来研究 T 细胞迁移的特定方面，例如对特定趋化因子信号的反应以及 T 细胞整合素与 ECM 结合蛋白之间的相互作用。该协议解决了分离和激活小鼠 CD8+ T 细胞的方法，包括二维空间中的 体外 迁移测定和用于分析指定 T 细胞迁移的计算分析工具。这些方法对用户是有利的，因为它们不需要复杂的材料或设备，就像文献中描述的其他一些细胞迁移测定一样。使用这些方法生成的细胞迁移数据可以以简单的方式提供免疫反应的证据，从而可以在 体内进行进一步的、明智的研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>15 mL conical tube</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>1x DPBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td>without calcium and without magnesium</td>
		</tr>
		<tr>
			<td>6 well plate non-TC treated</td>
			<td>Corning</td>
			<td>3736</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>FisherScientific</td>
			<td>352350</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>ACK lysing buffer</td>
			<td>ThermoFisher</td>
			<td>A1049201</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Allegra 6KR centrifuge</td>
			<td>ThermoScientific</td>
			<td>sorvall 16R with tx400 3655 rotor and bucket</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Beta mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>CellTrace Violet</td>
			<td>ThermoFisher</td>
			<td>C34571</td>
			<td>Or equivalent</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>ThermoScientific</td>
			<td>Sorvall ST 16R</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Collagen (IV)</td>
			<td>Corning</td>
			<td>354233</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>DeltaT culture dish .17 mm thick glass clear</td>
			<td>Bioptechs</td>
			<td>04200417C</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads Sheep anti-Rat IgG</td>
			<td>Invitrogen</td>
			<td>11035</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag 15 Magnet</td>
			<td>ThermoFisher Scientific</td>
			<td>12301D</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Easy sep mouse T cell isolation kit</td>
			<td>Stem Cell</td>
			<td>19851</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>SigmaAldrich</td>
			<td>F2442-500ML</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>SigmaAldrich</td>
			<td>10838039001</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td>http://fiji.sc/</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>Filter cubes</td>
			<td>Nikon or Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GK1.5</td>
			<td>ATCC</td>
			<td>TIB-207</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher</td>
			<td>15630080</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>HQ CCD camera</td>
			<td>CoolSNAP</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td>http://imagej.nih.gov/ij/h</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>ImageJ automatic tracking plug in</td>
			<td></td>
			<td>http://imagej.net/TrackMate</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>ImageJ manual tracking plug in</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/track/track.html</td>
			<td>weblink</td>
		</tr>
		<tr>
			<td>L-15</td>
			<td>Various</td>
			<td>See Materials</td>
			<td>Medium Recipe: Leibovitz&#8217;s L-15 medium without phenol red (Gibco) supplemented with 1-5 g/L glucose</td>
		</tr>
		<tr>
			<td>Liebovitz&#39;s L-15 medium, no phenol red</td>
			<td>ThermoFisher</td>
			<td>21083027</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lok disposable syringe</td>
			<td>Fisher Scientific</td>
			<td>14-955-459</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Lymphocyte separation medium</td>
			<td>Corning</td>
			<td>25-072-CI</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>M5/114</td>
			<td>ATCC</td>
			<td>TIB-120</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Microscope heating system</td>
			<td>Okolab</td>
			<td>okolab.com</td>
			<td>Custom designs available</td>
		</tr>
		<tr>
			<td>Millicell EZ slide</td>
			<td>Millipore</td>
			<td>C86024</td>
			<td></td>
		</tr>
		<tr>
			<td>Mojosort mouse CD8+ Na&#239;ve T cell isolation kit</td>
			<td>Biolegend</td>
			<td>480043</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse E-cadherin</td>
			<td>R&#38;D systems</td>
			<td>8875-EC-050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Mouse surgical dissection kit</td>
			<td>Fisher Scientific</td>
			<td>13-820-096</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>NIS elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>non-TC 24wp</td>
			<td>Corning</td>
			<td>353047</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Protein A</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>R9</td>
			<td>Various</td>
			<td>See Materials</td>
			<td>Medium Recipe: RPMI 1640x supplemented with 10 % FBS, 1 % antibiotic-antimycotic (Gibco), 20 mM HEPES buffer (Gibco), 1 % MEM Non-Essential Amino Acids (Gibco), 50 &#956;M &#946;-mercaptoethanol (Sigma-Aldrich)</td>
		</tr>
		<tr>
			<td>Recombinant mouse ICAM-1 Fc chimera</td>
			<td>R&#38;D systems</td>
			<td>796-IC-050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Recombinant Mouse IL2</td>
			<td>Biolegend</td>
			<td>575410</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>RPMI 1640x</td>
			<td>ThermoFisher</td>
			<td>11875093</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>T pins</td>
			<td>Fisher Scientific</td>
			<td>S99385</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>TE2000-U microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Various recombinant mouse chemokine</td>
			<td>R&#38;D systems</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>VCAM-1 Fc chimera</td>
			<td>R&#38;D systems</td>
			<td>643-VM-050</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Software</td>
		</tr>
	</tbody>
</table>

real-time in vitro migration assay for primary murine cd8+ t cells

适应性免疫反应依赖于 T 细胞在血液、淋巴和组织中迁移以响应病原体和异物的能力。T细胞迁移是一个复杂的过程，需要协调来自环境和局部免疫细胞的许多信号输入，包括趋化因子、趋化因子受体和粘附分子。此外，T 细胞运动受周围动态环境线索的影响，这些线索可以改变激活状态、转录景观、粘附分子表达等。 在体内，这些看似交织在一起的因素的复杂性使得很难区分有助于T细胞迁移的单个信号。该协议提供了从T细胞分离到计算机辅助分析的一系列方法，以在高度特定的环境条件下实时评估T细胞迁移。这些条件可能有助于阐明调节迁移的机制，提高我们对T细胞动力学的理解，并提供通过动物实验难以获得的强有力的机制证据。更深入地了解影响细胞迁移的分子相互作用对于开发改进的治疗方法非常重要。

可以通过流式细胞术确认 T 细胞活化，寻找 CD69 和 CD44 的表达增加，它们是小鼠 T 细胞活化的典型标志物6。此外，可以通过流式细胞术测定 CD3+ CD8+ T 细胞的 T 细胞群的纯度。该方法产生 >90% CD8+ T 细胞群。
T细胞迁移可以通过软件辅助细胞追踪程序进行评估，这些程序既可重复又可调，以满足研究者的需求。用于确定实验效果的一些?...

作者聚焦：通过实时分析 T 细胞迁移了解疾病机制

该协议提供了一种在特定环境条件下进行原代小鼠T细胞分离的方法和T细胞迁移的延时显微镜检查，并进行了定量分析。

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 ...

我们研究的目标是了解高度定义的环境条件下的 T 细胞迁移模式，以便进行明智的体外实验并破译影响迁移的分子相互作用。活体成像，如活体多光子显微镜，用于体内了解免疫细胞动力学。但微流体设备是一种常见的体外工具，可以对微环境进行精确控制，这在活体标本中是无法提供的。我们的方案使我们能够分析复杂的信号通路，并为靶向体外实验提供更好的理解，以细胞类型特异性的方式进一步解决单个信号的生物学作用。我们的方法简单、可重复，并且在具有明场显微镜和附加数码相机的研究实验室中易于使用。因此，我们的方法确保了一套可靠的方法来研究动态细胞迁移事件。我们的实验室将专注于研究免疫细胞的迁移，包括T细胞和中性粒细胞在各种疾病环境中的迁移，如肿瘤和感染，从而理解调节免疫细胞迁移的重要机制。这将有助于开发治疗方法，以帮助治疗和预防疾病。

real-time-in-vitro-migration-assay-for-primary-murine-cd8-t-cells

Research

JoVE Journal

Immunology and Infection

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

795 次观看.  University of Rochester. 我们研究的目标是了解高度定义的环境条件下的 T 细胞迁移模式，以便进行明智的体外实验并破译影响迁移的分子相互作用。活体成像，如活体多光子显微镜，用于体内了解免疫细胞动力学。但微流体设备是一种常见的体外工具，可以对微环境进行精确控制，这在活体标本中是无法提供的。我们的方案使我们能够分析复杂的信号通路，并为靶向体外实验提供更好的理解?...

视频: 作者聚焦：通过实时分析 T 细胞迁移了解疾病机制

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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

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

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

T细胞实时成像实时信号复合物的形成

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

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 ...

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 ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

小鼠边缘区 b 细胞剪切流诱导迁移的体外分析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

用于嵌合抗原受体 T 细胞的实时效力测定，用于定位固体和血液癌细胞

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

小鼠肾-居民CD8+T 细胞的分离，用于流细胞测量分析

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

一种基于DNA/Ki67的流式细胞术测定法，用于接种疫苗的小鼠抗原特异性CD8 T细胞的细胞周期分析

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.