感谢吴旭峰访问STORM显微镜。这项研究得到了国家癌症研究所（NCI）癌症研究中心和国家心肺血液研究所（NHLBI）的校内研究计划的支持。

注意:使用前请咨询所有相关材料安全数据表（MSDS）。本议定书中使用的几种化学物质是有毒和致癌的。执行协议时，请使用所有适当的安全措施，包括使用工程控制（通风橱，手套箱）和个人防护装备（安全眼镜，手套，实验室外套，全长裤，闭式脚趾鞋）。 1.活化T细胞复制成像<ol><li>使用Alexa 647抗体标记试剂盒与Alexa-647（A647）染料直接缀合抗体。遵循制造商提供的标签协议。 注意:在此步骤之前，建议在1x PBS中透析抗体溶液以提高抗体标记效率。 </li><li>在20,800 rcf下旋转标记抗体5 min，收集上清液以除去聚集的抗体。 </li><li> 荧光纳米金刚石涂层盖玻片的制备 <ol&gt; <li>用250μL的0.01％聚-L-赖氨酸（PLL）涂覆8孔盖玻片室（参见试剂/材料清单）15分钟，抽出并在65℃下干燥30分钟。 （干燥前不要冲洗） </li><li>在1x PBS中准备100 nm FND的稀释液。测试各种稀释液以确保在步骤1.2.7（我们使用制造商提供的FND的1:200稀释度）的每个视野中有足够的FNDs。 </li><li>涡旋稀释的FNDs 1分钟。 </li><li>在高功率设置下将FND上清液超声处理30秒。 </li><li>将超声处理的FND上清液在室温下在PLL涂覆的8孔盖玻片室中孵育30分钟。 </li><li>用1x PBS洗5次，并在TIRF显微镜上使用647nm激光激发可视化FND涂层室。理想情况下，如果在步骤1.2.2中对FND进行了适当的稀释，我们可以在视场中看到4-10个单独的FND（我们使用的是具有256 x 256个相机像素设置的100X物镜，以获得61 x 61μm的视野）与在在成像领域的每个象限中存在至少一个FND。如果FND似乎是聚类的（ 即 ，具有比周围FND显着更高的信号的FND和大于200nm的点扩散函数），则以6,800 rcf离心FNDs 1分钟，并使用FND上清液重复涂覆程序。 </li><li>在每个孔中加入250μl抗CD3抗体（10μg/ mL），并在37℃下孵育1小时，或在4℃下孵育过夜。 </li><li>取出溶液并加入1x PBS 30秒。重复此洗涤步骤5次（洗5次）。 </li></ol></li><li> 激活Jurkat T细胞 <ol><li>将1 ml Jurkat T细胞以800 rcf转染6 min，并重悬于300μl1x HBS溶液中。理想情况下，Jurkat T细胞在纺丝前的浓度应为0.5-1.0×10 6个细胞/ ml。 1 HBS溶液由前述22所述的HEPES缓冲盐水与1％牛血清白蛋白组成。 </li><li>加入150μL的每个孔中的1 HBS溶液，并在37℃孵育15分钟。 </li><li>向每个孔中加入50μL重悬浮的Jurkat T细胞，37℃孵育3分钟。 </li><li>向每个孔中加入300μL的4％对甲醛并在37℃下孵育30分钟。 </li><li>用1x PBS洗3次。 </li><li>通过在室温下加入250μL0.1％Triton-X溶液5分钟使细胞渗透。 </li><li>用1x PBS洗3次。 </li><li>在室温下向每个孔中加入250μL1％的在PBS中的1％鱼明胶溶液30分钟。 </li><li>用1x PBS洗3次。 </li></ol></li><li> 使用madSTORM成像激活Jurkat T细胞 <ol><li>在室温下向固定细胞中加入200μL0.1〜0.5μg/ mL的标记抗体1小时。 </li><li>用1x PBS洗5次。 </li><li>加入1ml的STORM缓冲液，并用玻璃盖玻片覆盖室，以限制暴露在空气中。之前已经描述了STORM缓冲区10 。我们建议马King新的STORM缓冲液，用于每轮成像，并将STORM缓冲液以20,800 rcf旋转2分钟以除去沉淀物。 </li><li>在TIRF模式下使用低647 nm激光功率，在视野中定位至少有3个FND的染色细胞。为了帮助定位FND，可以使用568 nm激光的伴随激励来提高FND信号的亮度。 注意:2.1节中描述的平均基准校正的性能随着更多的FND的增加而改善。 </li><li>增加647 nm激光功率并获取图像。我们通常以200 ms曝光，125 mW 647 nm激光，100X TIRF物镜（1.49 NA），100X EM增益（16位5 MHz，转换增益1））获取10,000帧。以前已经描述了我们的成像设置的细节10 。 </li><li>用1x TBS洗5次。 </li><li>加1 ml of洗脱缓冲液（3.5M MgCl 2，20mM PIPES，0.1％Tween-20，pH6.5），并在室温下孵育1分钟。 </li><li>重复洗脱3次。 （必须检查每种使用的抗体的精确洗脱条件和去除信号所需的洗脱漂洗次数）。 </li><li>用1x TBS洗3次，加入1ml 1x PBS。 </li><li>光漂白剂使用高功率（125mW）的647nm激光器，在405mW的405nm激光下以黑暗状态光活化A647染料。等待直到来自未洗脱的抗体的所有剩余信号被光漂白。通常这需要2-5秒的激光曝光。在洗脱和漂白后，使用1.3.5中的STORM设置获取样品图像（〜100帧），以确认信号的去除。 我们通常使用这种方法去除99.8％的信号。我们建议在madSTORM使用前测试每种抗体的洗脱效率，如前所述。 </li><li>加入250μL4％的多聚甲醛15 min。这个事件在细胞中固定分子的反向交联。 </li><li>用1x PBS洗3次。 </li><li>重复步骤1.3.1-1.3.12以顺序标记多个目标。 图1显示了使用madSTORM获得的激活的Jurkat T细胞中的多个分子的合成图像。 </li></ol></li></ol> 2.多路复用图像堆栈的漂移校正和对准<ol><li> 漂移校正的madSTORM图像堆栈 <ol><li>使用ImageJ打开图像。我们建议在ImageJ打开之前将图像文件转换为TIFF格式。使用矩形ROI来选择单个FND。播放时间流逝以确保FND在图像堆栈的每个帧中都可见。在ThunderSTORM插件中使用运行分析来定位FND。对于2.1.5中描述的平均基准校正方法，重要的是将单个FND定位在每个图像帧中（ 即确定的峰值数应为e等于帧数）。 </li><li>如果在每个帧中未识别到FND，请选择另一个FND。如果多个峰位于单个帧中，请删除偏离最早峰的峰。 </li><li>对图像堆叠中的每个FND重复2.1.2。请记住，漂移校正过程中不应包含一些FND，因此它们可以作为步骤2.1.7中所述的漂移校正精度的独立量规。 </li><li>为了更快，更精确的漂移校正，请使用ThunderSTORM中包含的互相关或基准校正算法来校正FND图像。通常，我们使用100个箱，5个放大倍率和0.1个轨迹平滑因子进行互相关，10个最大距离，0.1分钟标记可见度比和0.01个轨迹平滑因子进行基准校正。交叉相关和基准校正将产生一个可应用于其他FND的校正文件，以测试漂移校正精度。 </li><li>对于更严格，更精确的漂移校正，使用如前所述的平均基准校正（AFC）算法10 。该过程不需要优化参数，并且产生比SMLM定位算法预测的更高的定位精度。然而，平均漂移校正需要至少4个FND，并且将FND定位在图像堆栈的每个帧中。包含更多的FND，AFC漂移校正表现更好，因为大量的局部FND将减少给定帧中异常值峰值的失真效应。 </li><li>如前所述，使用ThunderSTORM对图像堆栈内的所有点扩散函数进行本地化。我们通常使用100nm的像素尺寸，每A / D计数为4.28的光电子，基准电平A / D计数为100，EM增益为100，PSF:集成高斯定位，5像素拟合半径，最大似然拟合方法和1.3像素初始西格玛</li><li>将从FND获取的漂移校正因子应用于整个图像堆栈的本地化。目视检查最终图像以确保正确的漂移校正（ 图2 ）。所产生的漂移校正因子应在AFC程序中未包含的FND上独立测试，以测量漂移校正精度的水平。漂移校正的独立FND的精度应接近从步骤2.1.6中使用的定位算法计算的平均精度/不确定度值。 </li><li>对于顺序标记的抗体的madSTORM图像堆叠重复步骤2.1.1-2.1.7。请记住，在每个madSTORM映像堆栈中对同一组FND进行本地化对于以下对齐过程至关重要。 </li></ol></li><li> 对齐madSTORM图像 <ol><li>计算每个madSTORM图像中漂移校正的FND的质心位置。要做到这一点，找到平均x和y轴po每个本地化FND的情景，并平均每个madSTORM图像中所有FND的平均x和y位置。以前已经描述了该步骤的详细算法10 。使用FND基准标记的质心位置对齐顺序的madSTORM图像。要执行对齐，通过从第一个madSTORM图像中减去第二个madSTORM图像中的FND的重心位置来计算两个本地化的madSTORM图像之间的偏移量。然后从第二个madSTORM映像中的所有本地化中减去偏移量。 </li><li>我们建议使用第一个madSTORM图像作为参考图像，并在第一和第三，第一和第四等之间重复此对齐步骤。通过测量未包括在对齐过程中的FND之间的偏移来测试对准精度。大的偏移可能表示在计算顺序的madSTORM图像的质心位置时使用不同的FND组。 </li><li>如果是这样，步骤2.2.1-2.2.2 shou使用同一组FND重复进行对齐。 注意:提供了AFC漂移校正和校准程序的自定义代码。 </li></ol></li></ol>

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我们没有什么可以披露的。

madSTORM中的顺序多路复用，漂移校正和校准程序允许在单元格中精确，高度多路复用异构结构的可视化。 10此外，madSTORM避免了多色STORM的局限性，如非远红色染料9,12的色差和次优照相切换/发射性能。由于洗脱步骤显着降低了来自顺序结合的抗体的空间干扰，所以可以使用madSTORM进行细胞样品的重复成像，超过先前技术9,15,21所达到的6-10轮复用成像。此外，顺序...

已经开发了各种超分辨率显微技术来克服光学显微镜（〜200nm）的衍射极限。其中包括一类称为单分子定位显微术（SMLM）的技术，包括光激活定位显微镜（PALM）和随机光学重建显微镜（STORM）。 SMLM技术共享使用可在on（荧光）和off（暗/光切换）状态之间切换的荧光团，允许来自单分子1,2,3的荧光的顺序定位。 由于其与商业上可获得的染料和显微镜的兼容性，STORM（dSTORM）直接成为广泛采用的SMLM技术。 dSTORM可以定期实现〜10 nm的定位精度，定义为计算衍射中心的不确定度离子限制点扩散函数（PSF）。然而，尽管使用本地化算法估计的高精度5,6,7 ，单个分子的实际位置的准确确定受到一些问题的阻碍。首先，在图像采集期间显微镜平台的机械运动增加了定位精度的显着不确定性。由于SMLM图像通过数千个延时帧获得，显微镜平台的纳米尺度运动可能会显着损害最终超分辨率图像8的精度。为了补偿图像采集期间的阶段移动，通常基于来自图像本身（互相关）或来自基准标记（基准校正）的顺序定位的基于回归的拟合来估计阶段漂移。 Howev呃，这些方法需要优化每个图像堆叠的多个参数，并且不能在诸如机械振动的短时间尺度上考虑阶段运动。金纳米颗粒和多色荧光珠已经用作SMLM中的基准标记，但它们不是光稳定的，并且在漂移校正后比使用的氮空位中心荧光纳米钻石（FND）显着降低精度在madSTORM 10 。 除了衍射极限外，光学显微镜进一步受光谱限制。多个目标的同时可视化需要具有非重叠光谱特征的荧光探针，通常将基于荧光的光学显微镜限制为6种颜色，并将SMLM限制为2-3色4,11,12。此外，非线性色差导致多色图像的偏移，w需要使用多色基准标记8,13的广泛对准程序。为了克服这些限制，先前的研究已经使用重复光漂白或连续结合的荧光团14,15,16,17,18,19的化学淬灭成像了多个靶。虽然这些方法可以克服显微镜的光谱极限，但是已知荧光漂白是有毒的方法20 ，而延长的漂白或猝灭可能引起不期望的副作用，例如交联失去。此外，荧光探针的积累可导致样品中结合位点的空间阻断，防止表位的大规模复用和强大的靶向。为了避免这种空间干扰，最近的一个小编使用随机交换自由扩散的蛋白质片段21实现多路复用。尽管这种方法允许细胞结构的密集标记，但是它需要广泛的生物化学制备以分离肽片段，不能定位单分子位置，并且不容易利用市售的探针进行大规模复制。我们提供一个详细的视频协议，描述荧光抗体的顺序结合和洗脱，用于复用，抗体大小限制的dSTORM（madSTORM）成像，以及使用荧光纳米金刚石来实现精确的漂移校正和对准。

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">8 well coverslip chamber</td>
			<td style="width:117px;">Lab-tek</td>
			<td style="width:124px;">155409</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% Poly-L-Lysine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NV-100nm Fluorescent Nano-diamond</td>
			<td>Adamas</td>
			<td>Red FND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD3&#949; antibody</td>
			<td>BD Biosciences</td>
			<td>555329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin, Fraction V</td>
			<td>KSE Scientific</td>
			<td>98-100P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Paraformaldehyde</td>
			<td>EMS</td>
			<td>15712</td>
			<td>Carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from fresh water fish skin</td>
			<td>Sigma-Aldrich</td>
			<td>G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647 antibody labeling kit</td>
			<td>Thermo Fisher</td>
			<td>A20186</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M7154</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cysteamine</td>
			<td>Sigma-Aldrich</td>
			<td>30070</td>
			<td>Highly toxic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclooctatetraene 98%</td>
			<td>Sigma-Aldrich</td>
			<td>138924</td>
			<td>Highly toxic, air sensitive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>KD Medical</td>
			<td>RGF-3210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x TBS</td>
			<td>KD Medical</td>
			<td>RGF-3385</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly multiplexed, super-resolution imaging of t cells using madstorm

使用单分子定位显微镜的成像异质细胞结构受到定位精度和复用能力不足的阻碍。使用荧光纳米金刚石基准标记，我们描述了在单分子定位显微镜中获得高精度所需的漂移校正和校准程序。另外，描述了新的复用策略madSTORM，其中使用荧光抗体的顺序结合和洗脱将多个分子靶向相同的细胞。在活化的T细胞上证明了madSTORM可视化在称为T细胞受体微团簇的膜结合的多蛋白结构内的不同组分的位置。另外，讨论了madSTORM作为多蛋白结构可视化的一般工具的应用。

使用顺序洗脱和染色方法在活化的Jurkat T细胞中产生微团簇和其他结构的多重madSTORM图像（ 图1 ，检查图对齐）。每个伪彩色图像表示在步骤1.1.1至1.3.13中获取的一轮madSTORM成像。如前所述10 ，应该对来自先前的非洗脱抗体的残留信号进行监视，以避免复用目标之间的串扰。此外，随着洗脱效率可以在抗体之间变化，顺序复用的顺序?...

Watch this Scientific Journal Video about Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM at JoVE.com

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

我们展示了使用荧光标记抗体的顺序结合和洗脱以单分子精确度在异质纳米结构内成像多个分子的方法。

使用madSTORM进行高分辨率，超分辨率的T细胞成像

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

highly-multiplexed-super-resolution-imaging-of-t-cells-using-madstorm

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

7.3K 次观看.  National Institutes of Health. 我们展示了使用荧光标记抗体的顺序结合和洗脱以单分子精确度在异质纳米结构内成像多个分子的方法。 

视频: Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

体外广角T细胞在小鼠淋巴结切片和共聚焦显微镜成像

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

扩展人外周血γδT细胞使用Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

从初级人类的幼稚和记忆T细胞的诱导调节性T细胞的生成

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

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

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 interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

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 ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

在从幼稚的CD4 + T细胞的人CD4 + FOXP3 +诱导调节性T细胞（iTregs的）使用含有TGF-β-协议的体外分化

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

使用成像流式细胞仪T细胞同种异体反应性测定

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...