D.M.O. acknowledges European Research Council grant No. 337187 and the Marie-Curie Career Integration Grant No. 334303. P.W.W. acknowledges the Natural Sciences and Engineering Research Council of Canada's Discovery Grant program.

注：阶段成像的前一天1和2来进行;确保前或上成像的天平衡到所使用的所有媒体和补充剂。平衡是通过媒体和补充变暖在培养箱设定为5％的CO 2来实现至37℃。所有细胞培养物的工作和盖玻片电镀步骤在无菌条件下在层流罩。 1.细胞转染<ol><li>转染T细胞进行成像24小时的实验用质粒编码荧光融合构建的F-肌动蛋白GFP标记和成像之前。注：细胞应分裂24小时，在转染前（48小时之前，成像），以确保细胞在最佳的健康，并在其对数生长期。 <ol><li>放置5毫升的RPMI补充剂（RPMI 1640 + 10％FBS和1％青霉素 - 链霉素（可选））在6孔板的单个孔进入孵化器中平衡。 </li><li>沉淀2×10 7个T细胞崇拜使用离心机管中于327×g离心5分钟，置的，在37℃5％的CO 2在RPMI补充和洗涤在预热的电介质的等体积。注：电穿孔介质是含有缓冲区和补充（见消耗品）降低血清培养基。 </li><li>后重新制粒在327×g离心5分钟，重悬细胞于500μl电介质，慢慢添加至试管中，添加5微克质粒的PCR级的dh 2 O用于标记F-肌动蛋白。 </li><li>轻轻拍打引擎盖上的工作区的反应杯，以确保去除任何气泡在媒体上，这可能会导致电力的电弧和向媒体内均匀地分布在细胞。 </li><li>打开电穿孔装置和设置电压300 V和电容290Ω。使用指数衰减为20毫秒电穿孔样品。注：可能需要不同的细胞和不同的质粒构建优化。 </li><li>一旦转染协议完成后，存储单元冰上继续之前1分钟。 </li><li>小心从试管转移细胞到从步骤1.1.1含有平衡的RPMI补充的5毫升的6孔板中。 </li><li>孵育细胞O / N在37℃+ 5％CO 2。 </li></ol></li></ol> 2.外套盖玻片和预暖成像补充<ol><li>外套盖玻片与抗体诱导T细胞刺激。 <ol><li>涂层的8孔盖玻片用1微克/毫升αCD3和αCD28在200μlPBS中的各腔室。 </li><li>储存在4°CO / N。可替代地，涂层盖玻片2小时之前，成像和保持在培养箱中在37℃至通过最大限度地减少温度变化到盖玻片在成像过程中减少漂移。 </li></ol></li><li>创建具有0.1μm的子衍射荧光微球盖玻片测试样品。 <ol><li>珠粒涡旋原液，以避免团块。 </li><li>在th的一种新的，干净的盖玻片e型中的那些相同步骤2.1.1使用，吸管5微升荧光微球原液到表面上（根据制造商的说明）。 </li><li>分布在与枪头盖玻片微球的解决方案。 </li><li>晾干，然后申请200微升安装介质如PBS。 注意：安装介质应与用于成像的细胞样品。 </li></ol></li><li>平衡的HBSS（+ 20mM的HEPES，以下称为“补充的HBSS&#39;）在一个离心管放置在培养箱中预暖O / N在37℃+ 5％CO 2。 注：加HEPES的是，如果使用的是孵化室的二氧化碳输入跳过此补充缓冲CO 2。 </li></ol> 3.显微镜建立<ol><li>打开TIRF-SIM显微镜和地方足够的蒸馏水的在显微镜的贮存阶段顶培养箱完全覆盖加热元件的基座。允许水以电子邮件quilibrate至37℃。加热套环添加到物镜，也设定为37℃。 注：由于在设置了灵敏度，这些步骤是以前做过的晚上，所以光学组件处于稳定的温度之前，成像。 </li><li>放置TIRF 488兼容光栅挡在显微镜的光路，并固定到位。 <ol><li>切换通道模式和光纤电缆连接到激光床和显微镜的阶段，“单”的位置分别从事的TIRF-SIM模式，正确的光路。注意：跳过此步骤会导致软件窗口内的错误（图1A）。 </li></ol></li><li>打开所有的显微镜和计算机部件，打开采集控制软件。 <ol><li>打开超频板，钛垫，N-SIM卡垫和ND采集窗口（ 图1B）。在“超频面板”中选择选项“TIRF 488&#39;（ 图2B，黄色箭头）。 </li&gt; <li>在N-SIM板窗口中，选择“TIRF-SIM”和“移动” 设置 （图2C，黄色箭头）。选择设置按钮 （图1C，黄色方框），然后选择“全内反射荧光488（用于100X TIRF 488nm的）&#39;从下拉菜单中，点击”应用“和”确定“。 </li><li>帧速率50毫秒，读出模式，EM增益3 MHz的14位和EM增益倍数300为1的转换增益（ 图1C，绿框）内的“DU897设置”来设置相机设置。 注：请参阅帧速率的考虑代表性的成果。 </li><li>通过选择100×1.49 NA CFI复消色差透镜的TIRF目的，对“N-SIM垫&#39;校准为TIRF-SIM显微镜的照明转动488纳米的激光功率为10％。 注：原始图像的像素大小为60纳米，与美景的512×512像素的领域。 </li><li>清洁使用镜头纸，以去除灰尘和油物镜。 </li></ol></li&gt; <li>找到TIRF-SIM焦平面。 <ol><li>开始与在尽可能低的z平面的目标。放置透镜油对目标的下降，并从步骤2.2到显微镜阶段添加荧光微球样品。 </li><li>选择位于显微镜的身体完美对焦系统（PFS）的功能。 </li><li>通过Z平面缓慢移动的目标，直到PFS按钮停止闪烁，以确认焦平面已经达到。 </li><li>切换到控制软件和图像的微球通过使用PFS微机械手轮做最后的调整“现场”视图。 </li></ol></li><li>观察均匀照明与离焦的无荧光以上75nm的渐逝波发射。 注意：确认结构照明通过观察从荧光微球的样品波动的照明模式实现。 </li><li>测量荧光微球的样品和量化改进成像resolut离子通过观察全宽的强度分布的半最大值（FWHM）。 <ol><li>打开分析软件（v4.20.01），安装了NIS-A N-SIM分析模块，并选择“应用程序” - &gt;“显示为N-SIM窗口”。 </li><li>与照明调制对比度（IMC）和高分辨率的噪声抑制（HRNS）设置为1的TIRF-SIM数据集（图2），重建微球体样本图像生成的1024×1024个像素的重建图像以30像素尺寸纳米。 </li><li>与1个像素（ 图3A中，黄色盒）的宽度使用线轮廓工具，通过一个单一的微球体的中心画线。 </li><li>选择将FWHM 按钮 （图3B，黄色盒），并点击在高斯分布的强度最大值，随后其强度最小量。 </li><li>确认微球体的FWHM是在100nm（图3C）的范围内。注：分辨率阿赫ieved成像当生物样本将取决于信噪比从标记效率和背景荧光变化。 </li></ol></li></ol> 4.样品制备成像<ol><li>盖玻片准备。 <ol><li>从冰箱或保温箱步2.1检索αCD涂层的盖玻片，并从每个孔取出PBS含αCD3和αCD28。 </li><li>从步骤2.3加入200μl预热HBSS补充到盖玻片的每个孔中。洗净并除去，以确保最小αCD3和αCD28留在溶液中。添加另一个200微升HBSS补充盖玻片井。 </li><li>清洁盖玻片的下侧与镜头纸以除去油和颗粒。 </li></ol></li><li>在显微镜台位置盖玻片并通过使用PFS z平面找到盖玻片TIRF焦平面如在步骤3.4中找到。 </li><li>细胞的准备。 <ol><li>吸取200微升媒体CON的泰宁转染的细胞从步骤1.1.8成微离心管中。 </li><li>沉淀细胞在微离心机中以268×g离心20秒并除去细胞介质。 </li><li>悬浮细胞在200μl来自步骤2.3预先温热的HBSS补充。 </li></ol></li></ol> 5.成像细胞<ol><li>取含细胞的HBSS补充到显微镜，轻轻移液加入200μl至孔中的一个。 </li><li>而在盖玻片（在PFS模式）留在TIRF焦平面，用显微镜目镜落射荧光照明跟踪荧光细胞接近盖玻片。 </li><li>一旦细胞土地上的盖玻片，切换到“现场”模式，在N-SIM垫带来的图像上的显示器，并检查细胞处于焦点的电脑屏幕上。 </li><li>要记录时间的课程，打开ND采集窗口，选择“时间”选项卡上，单击“添加”，并设置“间隔”为无延迟，“长”至10分钟，“环路”设置为1。 注意：持续时间可以根据所关注的方法来改变;当以“无延迟”捕获的STICS软件可以提取与≈10帧图像堆栈定量结果。 </li><li>以相同的采集设置到胎圈样品，首先在ND采集窗口中选择“立即运行”记录数据。 </li></ol> 6.重建SIM图片<ol><li>成像完成后，打开分析软件（v4.20.01），安装了NIS-A N-SIM分析模块，并选择“应用程序” - &gt;“显示为N-SIM窗口”。 </li><li>与照明调制对比度（IMC）和高分辨率的噪声抑制（HRNS）设置为1的TIRF-SIM数据集（图2），重建一个（或多个使用批量工具）原始SIM文件，以产生重构的SIM图像。 </li><li>确认重构文件具有30nm的像素大小，在所指示的图像状态栏。 注意：像素大小不影响STICS分析，但它应该是在尺寸比在PSF空间分辨率的至少2倍小，以确保像素之间的空间相关性。 </li><li>导出数据为.tiff文件，准备STICS分析。 </li></ol> 7. STICS分析<ol><li>下载并解压缩STIC（C）S Matlab软件包（怀斯曼实验室;可作为补充信息）。脚本stics_vectormapping.m是单通道STICS分析的核心和第一30行的代码被用作在手册的第2.1节中定义来定义的输入参数。打开这个脚本，并定义的输入参数TIRF-SIM卡的数据分析。建议也与子文件夹添加STIC（C）S文件夹复制到本地机器Matlab的路径，该手册的附录中详细说明。 <ol><li>通过编辑代码的相应行定义的输入参数的数据进行分析</li><li>要执行全日空的TIRF-SIM数据的裂解，以产生1向量地图，在第31行设置瞬时参数到颞分区域的帧的最大数量，并在管线32到1的时移为获得时间序列的矢量地图改变这些参数。 </li><li>对于不动去除过滤器，设置参数&#39;过滤&#39;（23行），以“移动平均”，并设置为21&#39;MoveAverage“参数（第24行）。 注意：该设置已被显示能用于移动较慢的和大的荧光信号，因为它是基于减去帧的串行数据帧被分析。 </li><li>更改线路33-38来设置输出文件和输出矢量地图的标题名称，以及输出目录路径，并确定以何种格式的矢量地图的图像应保存。 </li></ol></li></ol><table><tbody><tr><td>像素尺寸（微米） </td><td> 0.03微米</td><td>第19行</td></tr><tr><td>帧速率（秒） </td><td> 1秒</td><td>第20行</td></tr><tr><td>最大延迟时间（帧） </td><td> 20帧</td><td>第21行</td></tr><tr><td>分区域的大小（像素） </td><td> 8个像素</td><td>第29行</td></tr><tr><td>分区域的间距（像素） </td><td> 1个像素</td><td>第30行</td></tr></tbody></table><ol start="2"><li>打开在Matlab编辑器窗口中的脚本run1ChannelSTICS。通过运行线这个脚本的1-23加载图像序列。 注意：此脚本可用于加载和预处理图像系列，绘出矢量地图和速度直方图和运行批处理处理多个文件。如果需要的话，通过使用这个脚本的线24-26剪切图像的感兴趣的区域。 <ol><li>运行线路run1ChannelSTICS 29-35启动STICS分析。提示时，选择一个多边形来表示的感兴趣区域（ROI）的特定区域，如果需要的话。确保将ROI不与小区边缘重叠，边界伪影出现时STICS分析这些重gions。 </li><li>运行线路38-52，显示矢量地图。要绘制速度幅度直方图，运行线路53-72。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

该技术将允许研究人员的形象和以前无法解决的量化细节细胞表达荧光。在我们的结果，我们表明，F-肌动蛋白以对称方式从向小区中心中的T细胞是细胞周流动。这些发现与来自1D线轮廓kymograph数据13之前的结果一致，并延伸分析的能力，以二维和超分辨率数据。 所提出的技术是从kymograph成像它仅限于通过单个1D线随时间绘制荧光强度波动进展。在比较当前技术允许全...

该实验的目标是图像，并为了在免疫突触（IS）的形成建立流动方向和幅度表征filamentous-（F - ）的肌动蛋白的分子流在活的T细胞，超出衍射极限。这种技术将进行使用结构照明显微镜（SIM）中的全内反射荧光（TIRF）模式，成像的Jurkat T细胞上形成激活盖玻片包被有抗CD3-和抗CD28抗体的人工突触。 T细胞和目标之间的IS形式;抗原呈递细胞（APC）在T细胞受体（TCR）活化1,2。由于这是在确定是否适应性免疫系统产生要感染的免疫应答的第一步，不正确的响应性对刺激的后果可能导致许多疾病。的T细胞的APC相互作用的重要性是有据可查的，然而，初始TCR SI后成熟的作用（S）为gnal内化是尚未得到充分的理解。 作为细胞骨架被认为有助于两个链接到TCR活化（分子在质膜的移动和信号分子依赖于肌动蛋白骨架3,4的控制），了解如何细胞骨架重排的空间过程和时间上会阐明的步骤在突触形成的分子重组。 F-肌动蛋白逆行流动被认为是托起TCR集群对免疫突触的中心，这种易位被认为是信号停止和回收至关重要;辅助T细胞活化5的良好的平衡。 而标准荧光显微镜是一种灵活的工具，继续提供对多生物学事件，包括细胞 - 细胞相互作用的洞察力，它是由该系统的准确地解决多个荧光团位于比DIFFR接近能力的限制对方的行动限制（≈200纳米）。当细胞内的许多分子事件涉及的分子密集的人口，并表现出无论是在静止状态或在特定的刺激动态重配置，常规光学显微镜不​​能提供完整的图片。 采用超分辨率成像已经使研究人员能够绕过使用多种技术的衍射极限。单分子定位显微镜（SMLM）如PALM和STORM 6,7-必须解决分子的位置最多约20纳米的精度的能力，然而，这些技术依赖于长采集时间，建立一个图像在许多帧。通常这要求样品是固定的或对结构表现出低移动性，以便单一荧光团不误解为多个点。而受激发射损耗（STED）成像允许超分辨率通过非常有选择性的共焦激发8，所需的扫描方法可以是在视全细胞字段慢。受激发射损耗还演示显著光毒性更高的分辨率增加所致在耗尽光束的功率。 结构照明显微镜（SIM 9）提供了一个替代这些方法，能够在标准的荧光显微镜的分辨率增加一倍，并且是与活细胞成像比目前SMLM技术更兼容。它采用较低的激光功率，在一个宽视场系统的视图全细胞领域;提供更高的采集速度，并与标准荧光标记兼容。 SIM的宽视场的性质也允许全内反射荧光（TIRF），用于高选择性激励的利用率，以在&lt;100纳米的轴向尺寸的穿透深度。 图像相关光谱（ICS）是荧光强度相关的波动的方法。 ICS的演变;时空IM年龄相关光谱（STICS 10）相关的时间和空间内的荧光信号。通过关联像素强度与经由相关函数围绕滞后帧中的像素，将信息获得关于流的速度和方向。为了确保静态对象不与STICS分析干扰，不动的对象滤波器实现的，它的工作原理是减去像素强度的移动平均值。 这里提出的技术被认为是图像相关光谱的被施加到超分辨率显微镜数据，量化活细胞11的分子流的第一个示范。此方法对经由STICS分析12 F-肌动蛋白流衍射限制成像改进并会适合研究者谁希望确定二维分子流在活细胞中，从在超出光的衍射极限的空间分辨率获得的数据。

<table border="0" cellpadding="0" cellspacing="0" width="799">
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			<td height="20" style="height:20px;width:271px;">Consumables:</td>
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			<td height="20" style="height:20px;">Jurkat E6.1 cells</td>
			<td>APCC</td>
			<td>ATTC TIB-152</td>
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			<td height="20" style="height:20px;">RPMI</td>
			<td>Life Technologies</td>
			<td>21875-091</td>
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			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma</td>
			<td>F7524-500ML</td>
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			<td height="20" style="height:20px;">Penicillin-Strepromycin&#160;</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
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			<td height="20" style="height:20px;">HBSS</td>
			<td>&#160;Life Technologies</td>
			<td>14025-050</td>
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			<td height="20" style="height:20px;">HEPES</td>
			<td>Life Technologies</td>
			<td>15630-056</td>
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			<td height="20" style="height:20px;">#1.5 8-well Labtek coverslips</td>
			<td>NUNC, Labtek</td>
			<td>155409</td>
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			<td height="20" style="height:20px;">OptiMem</td>
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			<td>51985-042</td>
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			<td height="20" style="height:20px;">anti-CD3</td>
			<td>Cambridge Bioscience</td>
			<td>317315</td>
			<td></td>
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			<td height="20" style="height:20px;">anti-CD28</td>
			<td>BD Bioscience / Insight Biotechnology</td>
			<td>555725</td>
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			<td>165-2660</td>
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			<td height="20" style="height:20px;">Cuvette (0.4cm)</td>
			<td>BioRad</td>
			<td>165-2088</td>
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			<td height="20" style="height:20px;">Centrifuge (5810R)</td>
			<td>Eppendorf&#160;</td>
			<td>5805 000.327</td>
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			<td height="20" style="height:20px;">Centrifuge (Heraeus)</td>
			<td>Biofuge pico</td>
			<td>75003235</td>
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			<td height="20" style="height:20px;">6 well plate</td>
			<td>Corning</td>
			<td>3516</td>
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			<td height="20" style="height:20px;">Stage-top incubator</td>
			<td>Tokai Hit</td>
			<td>INUH-TIZSH</td>
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			<td height="20" style="height:20px;">Nikon N-SIM microscope</td>
			<td>Nikon</td>
			<td>Contact company</td>
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			<td height="20" style="height:20px;">Nikon Analyse software</td>
			<td>Nikon</td>
			<td>Contact company</td>
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			<td height="20" style="height:20px;">Incubator (190D)</td>
			<td>LEEC</td>
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</table>

cortical actin flow in t cells quantified by spatio-temporal image correlation spectroscopy of structured illumination microscopy data

丝状肌动蛋白起着在大多数细胞过程，包括运动，并在免疫细胞，被称为免疫突触的关键细胞间相互作用的形成至关重要的作用。 F-肌动蛋白还可以推测，以在细胞中包括子膜囊泡动力学和蛋白质聚类的膜调节分子分布发挥作用。虽然标准的光学显微镜技术，允许进行广义和衍射极限的观察，许多细胞和分子事件，包括集群和分子流发生在人群的长度尺度远远低于标准光学显微镜的分辨能力。通过全内反射荧光结合超分辨率成像方法结构照明显微镜，记录F-肌动蛋白中的T细胞的免疫突触二维分子流。时空图像相关光谱（STICS）随后被施加，其产生量化resulTS在速度直方图和代表流动方向性和大小的矢量地图的形式。这个协议描述的超分辨率成像和STICS技术相结合，以产生在细节的子衍射水平流矢量。这个技术被用于确认一​​个肌动蛋白的流动是对称逆行和向心力整个T细胞的周后突触的形成。

显微镜设置在成功地实现所有步骤研究者将具有F-肌动蛋白的流动是T细胞中突触的完成结构照明（SIM）成像（视频1），并量化这个分子流通过时空图像相关光谱（图4 11）。记录图像之前，确保样本照明是在9个原始图像均匀且在莫尔条纹在样品中观察到的形式，结构照明（图5），这些都是指?...

Watch this Scientific Journal Video about Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data at JoVE.com

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

cortical-actin-flow-t-cells-quantified-spatio-temporal-image

Research

JoVE Journal

Immunology and Infection

9.6K 次观看.  King's College London. To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy. 

视频: Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

性T细胞Transinfection从树突状细胞捕捉细菌

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

流式细胞仪用来评估染色质的T细胞国家

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

CD4的制备<sup&gt; +</sup&gt; T细胞通过显微镜分析GD3和GD2神经节苷脂膜表达的

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

由荧光相关光谱测量的一次淋巴细胞的细胞膜分子扩散

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...