我们感谢哥伦比亚大学生命科学研究所 (LSI) 成像设备, 用于支持和维护 STED 显微镜。这项工作由加拿大卫生研究所 (M.R.G.) 赠款 #68865 资助。我们感谢行藏 Kaibuchi 博士 (日本名古屋名古屋大学) CLIP-170-GFP 质粒。

所有动物程序均经英属哥伦比亚大学动物保育委员会批准。
1. A20 B 淋巴瘤细胞中 GFP 融合蛋白的表达
<ol><li>培养 A20 细胞完全 RPMI 培养基 (RPMI-1640 补充10% 热灭活胎牛血清 (血清), 50 µM 2-巯基乙醇, 2 毫米谷氨酰胺, 1 毫米丙酮酸, 50 U/毫升青霉素, 50 µg/毫升链霉素) 在37°c 在一个组织培养孵化器与5% CO2。</li>
	<li>根据制造商的说明, 准备转染试剂 (请参阅材料表)。 注意: 此协议已针对材料表中描述的特定试剂和转染协议进行了优化。我们尝试过的其他转染试剂没有产生足够的转染效率。</li>
	<li>使用 hemocytometer 计数单元格。离心机 2.5 x 106 A20 细胞 (为每转染) 5 分钟在 525 x g. 并用重悬了100µL 的细胞, 其中含有 1-2. 5 µg 的质粒 DNA。对于 CLIP-170-GFP23的 DNA 编码, 每转染使用2.5 µg。轻轻拌匀。</li>
	<li>将细胞转移到6井板的井中, 并将体积调整到2毫升, 预热完成 RPMI 培养基。培养细胞为18小时在37°c 允许蛋白质表达。</li>
</ol>2. 用 LPS 分离主鼠 B 细胞并激活它们
<ol><li>使用灭菌的外科工具从老鼠身上除去脾脏, 遵循该机构动物保育委员会批准的协议。</li>
	<li>在组织培养罩中, 将无菌70µm 细胞过滤器放入35毫米的组织培养皿中, 含有3毫升室温不育 PBS。使用5毫升注射器的活塞的橡胶部分, 通过细胞过滤器粉碎脾脏。</li>
	<li>吸管单细胞悬浮 splenoctyes 成无菌的15毫升管和离心机在 525 x g 5 分钟。</li>
	<li>使用磁性珠基 b 细胞隔离套件 (请参阅材料表) 以获得高度丰富的 b 细胞种群。</li>
	<li>并用重悬 b 细胞到 3 x106/毫升在完全 RPMI 培养基补充2.5 µg/毫升 LPS 加上 5 ng/毫升 B 细胞活化因子 (BAFF; 生存细胞因子)。</li>
	<li>培养细胞6小时在37摄氏度。</li>
</ol>3. 涂覆玻璃盖玻片抗玻璃抗体
<ol><li>准备抗体溶液来涂盖玻片。将山羊抗小鼠免疫球蛋白抗体的溶液稀释到室温无菌 PBS 中, 使其产生12.5 µg/毫升溶液;每盖玻片需要400µL 抗体溶液。 注: 对 A20 细胞使用山羊抗小鼠 IgG;对初级 B 细胞使用山羊抗鼠 IgM。山羊 IgG 抗体与小鼠 Fc 受体没有很好的结合。然而, 为了避免任何潜在的 Fc 受体绑定, 你可以使用 f (ab) 或 f (ab ')2片段的抗鼠 IgG 或抗鼠 IgM 抗体。</li>
	<li>蘸一 #1. 5 18 毫米圆玻璃盖玻片在100% 甲醇并且让它完全地烘干 (~ 10 分钟)。</li>
	<li>使用镊子, 将干盖玻片放入12井组织培养板和吸管400µL 12.5 µg/毫升抗体溶液 (5 µg 共计; 2 µg/cm2) 到盖玻片, 使其形成一个气泡在盖玻片的中心, 并蔓延到边缘。</li>
	<li>小心覆盖整个盖玻片, 但不允许抗体溶液延伸过去的玻璃盖玻片边缘和到组织培养塑料。室温孵育30分钟。</li>
	<li>用吹打1毫升的无菌 PBS 冲洗盖玻片, 随后将溶液吸出。重复两次以清除未绑定的抗体。</li>
	<li>在含有抗体涂层盖玻片的油井中加入1毫升的无菌 PBS, 直到细胞准备好添加。 注: 盖玻片可在室温下保存, 覆盖 PBS, 供同日使用。</li>
</ol>4. B 细胞在抗玻璃涂层盖玻片上的传播
<ol><li>制备改性 HEPES-缓冲盐水 (mHBS):25 毫米 HEPES, pH 值 7.2, 125 毫米氯化钠, 5 毫米氯化钾, 1 毫米 CaCl2, 1 毫米 Na2HPO4, 0.5 毫米 MgSO4, 1 毫克/毫升葡萄糖, 2 毫米谷氨酰胺, 1 毫米丙酮酸钠, 50 µM 2 巯基乙醇)。过滤器消毒此解决方案, 并存储在4摄氏度。</li>
	<li>在实验当天, 通过将1毫升的热灭活血清添加到50毫升 mHBS, 使50毫升的 mHBS 与2% 的血清 (mHBS)。在使用前将 mHBS 加热至37摄氏度。</li>
	<li>离心转染的 A20 细胞或原 B 细胞已培养的 BAFF 加 LPS 在 525 x g 5 分钟。</li>
	<li>并用重悬的细胞在1毫升的 mHBS, 计数的细胞使用 hemocytometer, 并稀释细胞到 2 x10 5 细胞/毫升在 mHBS-血清。</li>
	<li>使用镊子, 将盖玻片从组织培养板转移到石蜡膜上。</li>
	<li>将 B 单元格的250µL (5 x 104单元格) 添加到每个盖玻片。</li>
	<li>在黑暗中孵化出37摄氏度的盖玻片15分钟, 让 B 细胞在抗 IgG 涂层表面扩散。</li>
</ol>5. 固定和染色细胞
<ol><li>在室温蒸馏水中稀释16% 多聚甲醛溶液和50% 戊二醛溶液, 制备固定溶液, 最终浓缩3% 多聚甲醛和0.1% 戊二醛。在使用的当天准备固定液并丢弃任何多余物。 注意: 多聚甲醛和戊二醛库存解决方案只应用于化学油烟机。按照材料安全数据表 (MSDS) 的说明进行操作。</li>
	<li>准备通透/阻塞缓冲器 (3% BSA 和0.1% 海卫 X-100 稀释在 PBS)。过滤器消毒此解决方案, 并存储在4摄氏度。在实验当天, 将所需量加热至室温。</li>
	<li>准备染色缓冲剂 (PBS 中 1% BSA 和0.1% 皂苷)。使用0.2 µm 过滤器消毒此解决方案, 并存储在4摄氏度。在实验当天, 将所需量加热至室温。</li>
	<li>吸管350µL 固定溶液在每个盖玻片上, 确保表面完全覆盖。在室温下在黑暗中孵育10分钟。</li>
	<li>用 micropipet 从盖玻片的边缘小心地吸出, 从盖玻片中取出固定液。 注意: 应将固定液作为危险化学废料丢弃。</li>
	<li>用500µL 的通透/阻塞缓冲器清洗盖玻片。吸管的液体。</li>
	<li>Permeabilize 和阻断细胞通过增加250µL 的通透/阻塞缓冲到每个盖玻片, 并孵化10分钟室温下在黑暗中。</li>
	<li>稀释兔抗蛋白抗体1:100 在染色缓冲。</li>
	<li>从盖玻片中吸取通透/阻塞缓冲器, 并在每个盖玻片中添加50µL 稀释抗蛋白抗体溶液。在室温下在黑暗中孵育30分钟。</li>
	<li>在染色缓冲器中稀释532共轭山羊抗兔 IgG 二次抗体和 alexa 氟568共轭罗丹明 1:100, 制备二级抗体/肌动蛋白染色液。</li>
	<li>洗盖玻片3次, 通过添加500µL 的染色缓冲液来去除过量的原发抗体, 然后将其吸干。</li>
	<li>添加50µL 的二次抗体/肌动蛋白染色溶液的每盖玻片, 并在室温下孵化30分钟在黑暗中。</li>
	<li>清洗盖玻片3次, 通过添加500µL 的染色缓冲液来去除多余的二次抗体, 然后将其吸干。</li>
	<li>将10µL 的安装试剂添加到显微镜滑梯上。将盖玻片安装到显微镜下的幻灯片上, 单元格侧向下。 注: 强烈建议使用 "防褪色" 安装介质 (请参阅材料表) 以保持 GFP 融合蛋白的荧光强度。避免使用含有 DAPI 的安装试剂, 在使用 STED 激光时会干扰显影的成像。</li>
	<li>允许幻灯片在黑暗中过夜。第二天图片幻灯片。 注: 强烈建议在盖玻片干燥后对幻灯片进行成像, 因为 GFP 荧光会随着时间的推移而减少。</li>
</ol>6. 用 STED 显微镜成像
注意: 请注意下面描述的所有软件步骤都是特定于我们使用的显微镜和软件 (请参阅材料表)。如果使用不同的显微镜/软件执行成像, 则需要调整步骤和设置。
<ol><li>打开 STED 显微镜, 激活激光器和荧光照明灯, 并启动显微镜软件。</li>
	<li>设置 STED 损耗激光器的参数。 注意: 这将取决于所使用的特定显微镜和它所配备的激光器。一些推荐的设置是白光 (70%);592毫微米激光 80%;660毫微米激光80%。</li>
	<li>使用100X 目标激活 STED 设置并对齐 STED 激光光束。</li>
	<li>使用目镜, 聚焦样品, 并选择一个有适度 GFP 表达式的细胞。 注意: 低 GFP 表达式将不可见, 因为 STED 降低了发射强度。相反, 高表达 CLIP-170-GFP 诱导大簇的自发形成, 不反映正常微管和端蛋白复合体的形态和分布。选择具有适度 GFP 表达式的细胞对于准确地成像抗原接触部位的骨架结构至关重要。</li>
	<li>缩放到所选单元格, 然后选择要成像的感兴趣区域。调整焦点, 使最接近盖玻片的 B 单元格的x-y 平面处于焦点。<ol>
<li>要做到这一点, 将目标逐步接近盖玻片, 使 B 细胞骨架结构成为焦点, 然后走出焦点。然后逐步将目标转向相反的方向, 直到 B 细胞骨架结构重新回到焦点。</li>
	</ol>
</li>
	<li>优化设置, 包括激光功率, 励磁光束和探测器范围。从制造商的说明推荐的设置开始。然后根据需要进行调整以优化样品的信号。将所有示例的图像与相同的设置进行比较。</li>
	<li>在软件的 "获取" 选项卡下, 通过检查 "帧之间" 的选项, 将图像捕获设置为在左下 "顺序扫描" 面板中的序列帧获取。</li>
	<li>设置捕获序列。 注意: 我们首先获取 gfp 荧光, 以避免 gfp 信号的降解。 根据除 GFP 之外使用的显影的组合, 成像较长波长荧光信号首先可能产生更好的结果。然而, 显影或荧光蛋白受到刺激的排放损耗和成像的顺序应进行优化, 以获得最强的荧光信号和最佳分辨率。</li>
	<li>在软件的 "获取" 选项卡下选择并设置每个荧光的 STED 激光功率, 并使用 592 nm 或 660 nm STED 激光器的滑块来调整激光功率。调整荧光蛋白和 Alexa 532 的 592 nm 损耗激光器的功率。调整 660 nm 损耗激光器的功率为 Alexa 568。</li>
	<li>要增加分辨率并减少背景信号, 请转到软件 "获取" 选项卡下的 "获取" 设置, 通过选择每个选项的下拉菜单, 选中大于1的值, 增加行和/或帧的平均值。</li>
	<li>此外, 通过检查 "获取" 选项卡下的荧光的浇口选项, 并指定时间门的值 (见下文附注), 使用时间门控 STED 获取荧光信号。增加 STED 激光功率来提高分辨率, 虽然这也会增加显影的漂白。 注: 需要对所使用的显微镜、样品和显影进行优化。建议使用0.3 到 6 ns 的时间门。在固定后, GFP 对高激光功率特别敏感。为了减少 GFP 的漂白, 应应用时间门, 减少 STED 激光功率。</li>
	<li>要捕获 3D STED 图像, 请使用滑块将点传播函数 (PSF) 更改为 "3D STED"。</li>
	<li>手动获取多个图像以确保重现性。使用反卷积软件包41Deconvolve 图像 (参见图 1) (请参阅材料表)。</li>
</ol>

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作者没有什么可透露的。

骨架结构的详细图像可以使用 STED 超分辨率显微镜获得, 理论上可以达到 50 nm 的分辨率, 与传统的共焦显微镜相比, 衍射有限分辨率为 200 nm。24. 利用反褶积软件计算原始光源从观测到的 "模糊" 荧光信号中最可能的位置, 进一步提高了解析精细结构的能力。本协议描述了使用 STED 图像肌动蛋白和微管骨架以及细胞骨架相关蛋白的方法。
B 细胞活化诱发肌动蛋白细胞骨架和微管网络的重塑, 两个骨架的协调调节对免疫突触形成有重要意义17,42。我们提出的方法已被优化, 以同时成像的肌动蛋白和微管骨架在抗原接触部位使用多色 STED, 但同样适用于单色 STED。先前我们对 B 细胞细胞骨架进行的研究表明, STED 可以为细胞结构的组织及其相互作用提供新的见解。使用共焦和全内荧光 (TIRF) 显微镜, 我们观察到, 微管接触的 F 肌动蛋白环的周围的抗原接触点17。使用 STED 显微镜, 我们能够表明, 由 + 尖端标记的微管的加端 CLIP-170 与细胞外围的树突肌动蛋白网络紧密联系在一起 (请参见图 4)。
多因素影响哪一种成像技术最适合自己的具体应用。这些方法包括所需的分辨率、要成像的结构、标记技术及其信噪比 (即、对比度)、采集时间、样品制备的简便性和重现。STED 的样品制备方法与共焦显微术相比没有显著的差异, 它结合了高分辨率和快速图像采集。STED 的一个主要优点是, 它是一个光学过程, 其中图像直接从样本获取, 分辨率可以通过改变 STED 激光器的功率24进行调整。与地面状态损耗超分辨率显微术不同, 从数以千计的连续图像捕获重建图像, STED 不需要广泛的计算处理, 并避免了图像重建工件的引入24. 但是, STED 图像中的对比度通常是低24, 在这种情况下, 可能需要使用诸如 ImageJ 之类的软件进行后映像处理以增强对比度。这对于具有致密结构的图像尤为重要, 如树枝状肌动蛋白网络。为了提高图像采集过程中的图像对比度, 可以减少激光功率损耗和/或应用直线或帧平均。时间门控 STED 在用户设定的时间延迟后捕获光子, 通过减少收集光子的区域 (2443), 可以提高分辨率。我们建议在免疫突触中优化骨架结构的 STED 成像, 结合这些方法提高对比度和分辨率。
目前, 不是所有的显影都是最佳的成像与 STED, 而不是所有的荧光组合适合获得多色 STED 图像。仔细调整检测范围对于确保显影到相邻通道的最小出血量非常重要。该协议中使用的显影组合 (即、GFP、alexa 532 和 alexa 氟 568) 是对多色 STED 超分辨率成像的最佳选择。与结构化光照显微镜 (SIM) 和单分子定位方法 (SMLM) 相比, 光激活定位显微镜 (PALM), STED 通常不是理想的多色成像。然而, 我们在这里展示了荧光检测的轻微过度饱和, 与简单的图像处理工具配对, 可以提供高分辨率的肌动蛋白和微管骨架的多色图像。
骨架结构的 STED 成像协议揭示了 B 细胞免疫突触的骨架结构的新细节。尽管我们对 B 细胞抗原接触部位的肌动蛋白和微管骨架进行了优化, 但这些方法应适用于其他细胞类型, 特别是免疫细胞 (T 细胞、NK 细胞、肥大细胞、等), 形成免疫突触。此外, 该协议的效用可以扩展到涂层盖玻片与其他配体或粘合剂基板。然而, 优化协议和图像采集设置对于细胞类型和实验设置是非常重要的。

当 b 细胞绑定到抗原呈现细胞 (apc) 表面上的偏振光阵列 (例如) 时, 由此产生的 b 细胞受体 (BCR) 信号驱动了典型的免疫突触结构的形成, 这首先描述在T 单元格1,2,3,4,5,6,7,8,9,10, 11,12,13。最初, microclusters 抗原束缚 BCRs 形成在 B 细胞的外围: APC 接触点。这些 microclusters 然后移动到抗原接触点的中心, 在那里他们合并成一个中央超分子活化星团 (cSMAC), 形成了免疫突触的核心。免疫突触形成优化 BCR 信号和促进 BCR 介导的抗原提取从 APC 膜14。这种抗原的获取, 其次是 BCR 介导的抗原内化和随后的抗原处理, 允许 B 细胞呈现肽: MHC II 配合物 t 细胞和诱导 t 细胞帮助14。由于免疫突触形成促进 B 细胞活化, 阐明建立这种功能模式的受体组织的机制可以为如何启动和调节体液免疫反应提供新的见解。
重组肌动蛋白和微管骨架是必不可少的免疫突触形成。由空间极化的抗原引起的局部 BCR 信号诱导迅速和戏剧性地重塑肌动蛋白骨架1,15。b 细胞周围的树突状肌动蛋白结构的形成, 推动了细胞膜的作用力, 促进了 b 细胞的扩散。这使得 B 细胞可以扫描更大面积的抗原轴承表面, 并增加 BCRs 的数量, 绑定抗原和激活 BCR 信号通路。同时, MTOC 和微管网络被重新定向到抗原接触部位。当 MTOC 接近抗原接触点时, 从 MTOC 中产生的微管沿等离子体膜的内表面延伸, 在 B 细胞和抗原轴承表面之间的界面上16,17。这些 juxtamembrane 微管可以充当蛋白介导的 BCR microclusters18的向心运动的轨道, 导致 cSMAC 的形成。
MTOC 对免疫突触的重新定位和极化需要完整的肌动蛋白和微管骨架, 并且通常取决于皮质肌动蛋白网络和微管的相互作用16,17, 19,20。皮质肌动蛋白结合蛋白, 如 IQGAP1, 可以通过与修饰微管加末端21的蛋白质复合物相互作用来捕获微管。这些附加端结合蛋白的动态复合体包括 EB1 和 CLIP-170, 统称为微管加端跟踪蛋白 (+ 提示)21,22。+ 微管末端的提示可以绑定到与血浆膜或皮质肌动蛋白细胞骨架相关的蛋白质。这允许产生力机制 (例如, cortically 锚蛋白沿微管的负端定向运动) 在微管上施加拉力, 从而重新定位 MTOC。CLIP-170 可以绑定到肌动蛋白相关的支架蛋白 IQGAP123, 我们已经表明, 这两个蛋白质是需要 BCR 诱导 MTOC 极化向免疫突触17。这种 IQGAP1-CLIP-170 的相互作用可能在协调肌动蛋白细胞骨架的重塑和微管网络在 B 细胞免疫突触的重新定位方面发挥关键作用。
常规荧光显微镜揭示了肌动蛋白和微管骨架在 B 细胞免疫突触形成中的戏剧性重组2。然而, 由于光的衍射极限, 这种方法无法详细解决小的蜂窝结构, 根据阿贝定律, 它依赖于用来照亮样品的光波长和目标24的光圈。这个衍射极限限制常规光学显微镜的分辨率到200-300 毫微米在横向方向和500-700 毫微米在轴向方向25。因此, 更小的亚细胞结构, 以及肌动蛋白和微管骨架的细微细节, 只能通过电子显微镜观察。细胞骨架的电子显微成像是耗时的, 需要苛刻的标本固定和准备协议, 可以改变生物结构, 并限于抗体介导的检测。immunostain 和同时图像多蛋白或细胞结构的能力是荧光显微镜的一个显著优势。此外, 在细胞中表达荧光融合蛋白能够实时成像, 并且在染色的有效抗体无法获得的情况下是有用的。
最近在超分辨率显微镜技术方面的进展克服了光的衍射极限, 允许纳米蜂窝结构的可视化24。一种超分辨率显微技术被称为受刺激的排放损耗 (STED) 显微镜。STED 使用两个激光器, 其中一个激光激发荧光和一个甜甜圈形状模式的第二个激光器选择性地抑制荧光周围的荧光发射。这减少了单个荧光粒子的点传播函数 (表观面积), 并提供了一个亚衍射极限荧光图像25,26。地面状态损耗显微镜还采用荧光技术获取超分辨率图像。然而, 图像的采集和重建时间长, 只有有限数量的显影, 可以使用, 同时高分辨率成像多骨架组件是技术上的挑战, 因为保持肌动蛋白和微管结构需要不同的固定程序。因此, STED 在电子显微术和其他超分辨显微学方法方面具有多种优势, 因为它提供了快速的图像采集, 具有极少的后处理要求, 并采用相同的显影和染色技术。用于固定样本的常规荧光显微术26。
超分辨率显微术现已被用于可视化肌动蛋白结构的免疫突触在自然杀手 (NK) 细胞和 T 细胞26,27,28,29,30,31. 然而, 微管细胞骨架的超分辨成像, 以及免疫突触形成过程中肌动蛋白和微管骨架的协调重组, 最近才被报道为17。我们使用 STED 显微镜的图像 B 细胞, 已获准在盖玻片涂上抗免疫球蛋白 (抗组织) 抗体, 刺激 BCR 信号和启动细胞骨架重组。在固定化的抗球蛋白抗体的镀膜下, B 细胞经历剧烈的肌动蛋白依赖性传播, 这概括免疫突触形成过程中的初始事件。重要的是, STED 显微镜揭示了在免疫突触周围形成的 F 肌动蛋白的树突环的细微细节, 并表明 MTOC, 以及附着于它的微管, 已经靠近抗原接触点17。这些微管向外延伸向 F 肌动蛋白的外围环。此外, 多色 STED 成像的各种组合的 F 肌动蛋白, 蛋白, IQGAP1 和 GFP 标记 CLIP-170 + 提示表明, 微管加端标记 CLIP-170-GFP 与周围肌动蛋白网和 IQGAP1 密切联系,皮质捕获蛋白17。
在这里, 我们提出了一个详细的协议, 以成像肌动蛋白和微管骨架在免疫突触使用 STED 显微术。利用 A20 小鼠 B 细胞系对这些方法进行了优化, 广泛用于研究 BCR 信号和免疫突触形成17,32,33,34,35,36,37,38,39. 由于 CLIP-170 的商业抗体在以前的实验中对染色没有很好的效果, 我们详细描述了 A20 细胞中 GFP 标记 CLIP-170 的表达, 以及同时可视化的染色协议, 以三骨架成分或细胞骨架相关蛋白。在 NK 细胞免疫突触中使用 STED 显微术的图像肌动蛋白的方法已经被描述了以前的40。在这里, 我们扩展到获取多色超分辨率图像的肌动蛋白和微管骨架在 B 细胞。
超分辨率显微镜的一个关键考虑是使用适当的固定程序来维持细胞结构和防止荧光蛋白的损伤。本文提出的固定和染色方法已被优化, 以保留 GFP 荧光和提供高分辨率成像的肌动蛋白和微管网络。在表达荧光蛋白时, 应该注意到 B 细胞通常很难染。使用此协议, 20-50% 的 A20 细胞通常表达转染的 GFP 融合蛋白, 在这个人群中蛋白质表达的水平是可变的。然而, 使用我们描述的程序, 超分辨率成像的肌动蛋白和微管是相当健壮的, 高质量的图像是容易获得的。尽管它们的尺寸相对于 A20 细胞较小, 但我们表明, 这些程序也可以用于图像的微管网络在初级 B 细胞已短暂激活的脂多糖 (LPS)。我们已经表明, 脂多糖活化的初级 B 细胞可以在相对较高的效率 (即, 这样的蛋白质消耗可以检测到免疫印迹法) 转染 siRNAs, 使其成为一个良好的替代使用 B 细胞线的一些研究17。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A20 mouse B-lymphoma cells</td>
			<td>ATCC</td>
			<td>TIB-208</td>
			<td>Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td>Heat inactivate at 56 oC for 30 min</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Millipore Sigma</td>
			<td>G5763</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Millipore Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Liquid, 10,000 units</td>
		</tr>
		<tr>
			<td>Additional materials for primary B cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bead-based B cell isolation kit</td>
			<td>Stemcell Technologies</td>
			<td>19854</td>
			<td>EasySep Mouse B cell Isolation kit</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L4391</td>
			<td>LPS from E. coli 0111:B4</td>
		</tr>
		<tr>
			<td>B cell-activating factor (BAFF)</td>
			<td>R&#38;D Systems</td>
			<td>2106-BF-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding CLIP-170-GFP</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Kozo Kaibuchi (Nagoya Univ., Nagoya, Japan); described in Fukata et al. Cell 109:873-885, 2002</td>
		</tr>
		<tr>
			<td>Recommended transfection reagents and equipment </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Nucleofection kit V</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>Follow the manufacturer&#39;s directions for mixing the transfection reagents with the DNA</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector model 2b</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program L-013 used</td>
		</tr>
		<tr>
			<td>Falcon 6-well plates, TC treated, sterile</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Coating coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18-mm diameter round #1.5 cover glasses</td>
			<td>Thomas Scientific</td>
			<td>1217N81</td>
			<td>Similar product: Marienfield-Superior catalogue #117580</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>1381242</td>
			<td>Dissecting extra fine splinter forceps</td>
		</tr>
		<tr>
			<td>Falcon 12-well sterile tissue polystyrene tissue culture plate</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>100% methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile phosphate buffered saline (PBS) without calcium or magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-008</td>
			<td>For A20 cells</td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgM antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td>For primary mouse B cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% stock solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (50% stock solution)</td>
			<td>Millipore Sigma</td>
			<td>340855</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Millipore Sigma</td>
			<td>S2149</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), Fraction V</td>
			<td>Millipore Sigma</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab52866</td>
			<td>1:250 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 532-conjugated goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11009</td>
			<td>1:100 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td>1:100 dilution recommneded but should be optimized</td>
		</tr>
		<tr>
			<td>Prolong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td>Without DAPI</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>P1150-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Millipore Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 TCS STED microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens deconvolution software</td>
			<td>Scientific Voume Imaging</td>
			<td></td>
			<td>See https://svi.nl/HuygensProducts</td>
		</tr>
	</tbody>
</table>

visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy

b 细胞与膜结合抗原 (例如, 在抗原呈现细胞的表面) 形成免疫突触, 一种专门的细胞结构, 优化 B 细胞受体 (BCR) 信号和 BCR 介导的抗原获取。肌动蛋白细胞骨架的重塑和微管网络对抗原接触部位的重新定位是免疫突触形成的必要条件。将肌动蛋白细胞骨架重塑为 F 肌动蛋白的致密外周环, 伴随着微管组织中心向免疫突触的极化。微管加端结合蛋白, 以及皮质加端捕获蛋白介导肌动蛋白和微管骨架之间的物理相互作用, 使它们能够以协调的方式进行重组。阐明控制这种骨架重组的机制, 以及了解这些骨架结构如何形成免疫突触生成和 BCR 信号, 可以为 B 细胞活化提供新的洞察力。这已经得到了发展的超分辨率显微方法, 揭示了新的细节骨架网络组织。这里我们描述了一种使用受刺激的排放损耗 (STED) 显微镜同时图像肌动蛋白结构, 微管和转染的 GFP 标记微管加端结合蛋白在 B 细胞中的方法。为了模拟免疫突触形成的早期事件, 我们允许 B 细胞在盖玻片上涂上抗免疫球蛋白 (抗组织) 抗体, 启动 BCR 信号和细胞骨架重塑。我们提供了一步一步的协议, 以表达 GFP 融合蛋白在 A20 B 淋巴瘤细胞, 抗细胞扩散, 并为后续的细胞固定, 染色, 图像采集和图像反褶积步骤。使用这些程序获得的高分辨率图像使人能够同时可视化肌动蛋白结构、微管和微管加端结合蛋白, 这两种骨架网络之间可以连接。

对于在固定化抗 STED 上传播的 B 细胞, 与反褶积软件结合使用的显微术提供了比共聚焦显微镜更高的骨架结构分辨率图像。这在图 1中很明显, 其中 F 肌动蛋白网络是使用上面描述的协议进行可视化的。对同一样本的共焦和 STED 超分辨率图像的比较表明, STED 图像具有较高的分辨率, 揭示了肌动蛋白骨架的更详细结构 (图 1)。这一数字还表明, 反褶积是获得高质量的 STED 图像, 其中肌动蛋白花丝更明确的定义是必不可少的。尽管共焦图像的反褶积在图像分辨率上产生了显著的改善, 但 deconvolved STED 图像提供了比 deconvolved 共焦图像更详细的结构信息。特别是, STED 显微镜 (图 1和图 2) 更详细地揭示了外围 F 肌动蛋白环的树突状结构。抗原接触点的微管网络也使用上面描述的样本准备和成像协议 (图 3) 进行成像。微管起源于一个中心点, 即 MTOC, 向外向细胞的外围散发。在这个实验中, B 细胞被允许扩散到抗涂布盖玻片15分钟(图 3), MTOC 已经移动到抗原接触点17 的时间点。在图 3中显示的微管的末端可以看到标记微管的加号的 CLIP-170-GFP 簇。当微管网络的样品制备和 STED 成像最佳时, 观察到连续和明显的微管, CLIP-170-GFP 沿微管或加端局部定位 (图 3A)。亚优微管染色, 这是观察当使用较低浓度的染色抗体或批次的α蛋白抗体, 超过一岁, 导致微管出现作为不连续的部分, 是很差解决在反褶积 (图 3B; 另请参见图 5C)。尽管这些图像中的所有 CLIP-170-GFP 荧光都与α-蛋白-immunostained 结构相关联, 但由于不完整的染色, 人们无法区分 CLIP-170-GFP 是位于加端, 还是沿微管长度。微管的。因此, 必须将α蛋白抗体储存在制造商推荐的贮存条件下, 并在一年内使用。
使用此协议, 高质量的多色 STED 图像, 显示肌动蛋白细胞骨架和微管网络的组织和结构, 以及与这两个骨架17关联的 IQGAP1 和 CLIP-170 的蛋白质,可以获得。图 4中的 STED 图像显示了树突肌动蛋白的外围环, 以及在肌动蛋白染色不太密集的细胞中的中心位置发出的微管。这些微管末端的 CLIP-170-GFP 与周围的 F 肌动蛋白紧密相关。该协议允许一个使用单色 STED 成像 (图 2) 或多色 STED 成像 (图 3和图 4) 在免疫突触中可视化骨架结构。但是, 应该注意的是, 单色 STED 成像 (图 2) 可能比多色 STED (图 4) 更好地解析 B 细胞中肌动蛋白结构和微管。这可能是由于不同显影序列 STED 图像采集所引起的漂白。为了获得最佳的超分辨率图像, 显影和荧光蛋白的组合, 以及它们使用励磁和损耗激光器进行成像的顺序, 应该对样品进行优化。然而, 多色 STED 成像提供了更高的分辨率图像, 这些骨架结构比传统的共焦显微镜。此外, 单色 STED 可用于获取整个 B 细胞肌动蛋白或微管网络的3维超分辨率图像 (影片 1)。
当使用与荧光融合蛋白转染的细胞时, 达到最佳表达水平并避免因过度表达而导致的伪影是重要的考虑因素。在 CLIP-170-GFP 为抗原的单元格中, 形成 CLIP-170-GFP 的大聚合体 (图 5A)。除了此 mislocalization 的 CLIP-170-GFP 外, 此单元格中仅有一部分微管网络位于与盖玻片最近的焦点平面(图 5B) 中。这表明 CLIP-170 过度表达也可能损害 BCR 诱导的 MTOC 极化。相反, 由于强烈的荧光信号通常需要获得高质量的 STED 图像, 低表达的荧光融合蛋白, 如 CLIP-170-GFP (图 5C) 导致质量较差的图像。因此, 当使用已转染荧光蛋白的细胞时, 重要的是只对那些具有最佳融合蛋白表达水平的细胞进行图像处理。还必须注意的是, 用于 A20 单元格的转染协议 (请参见材料表) 通常会导致20-50% 的细胞表达所染蛋白。对于质粒 DNA (相对于 siRNAs), 主 B 细胞的转染频率通常比 A20 细胞低得多, 需要使用 B 细胞线。但是, 可以使用此协议 (图 6) 获得 untransfected 主 B 单元格中骨架元素的高质量 STED 图像。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57028/57028fig1.jpg"> 图 1: F 肌动蛋白的共焦和 STED 成像的比较.共焦图像 (顶部) 和 STED 图像 (底部) 的 A20 细胞已蔓延的抗 IgG 涂层盖玻片15分钟之前, 被染色的 Alexa 氟532共轭罗丹明。使用共焦 STED 显微镜, 同一细胞首先通过共焦显微镜成像, 然后由 STED。首次共焦和 STED 图像显示, 连同相同的共焦和 STED 图像反褶积。刻度条: 5 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/57028fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57028/57028fig2.jpg"> 图 2: 抗原接触部位的肌动蛋白细胞骨架.A20 细胞被允许在抗 IgG 涂层的盖玻片上传播15分钟, 染色了 Alexa 的568共轭罗丹明, 并通过 STED 显微镜进行成像。最初的 STED 图像是 deconvolved 的。面板A B和面板-c显示两个不同单元格的代表性图像。面板E显示了面板C中白色框中区域的3X 放大。面板-d的缩放栏: 5 µm 面板E: 1 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/57028fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57028/57028fig3.jpg"> 图 3: 微管网络和 CLIP-170-GFP 的 STED 图像.A20 细胞表达 CLIP-170-GFP 被允许扩散到抗 IgG 涂层盖玻片15分钟之前被固定和 immunostained 与α蛋白抗体加上 Alexa 氟532共轭二级抗体。(A) 代表性图像, 显示微管网络的染色, CLIP-170-GFP 主要位于微管的加号端。(B) 由于使用过期的α蛋白抗体, 所以微管的次最佳染色和分辨。刻度条: 5 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/57028fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57028/57028fig4.jpg"> 图 4: 肌动蛋白和微管骨架的 STED 图像.A20 细胞表达 CLIP-170-GFP 被允许扩散到抗 IgG 涂层的盖玻片15分钟之前被固定和染色的 Alexa 氟568共轭罗丹明可视化 F 肌动蛋白, 并与α蛋白抗体加上 Alexa 流体532共轭二次抗体可视化微管。面板-d和面板E F显示两个不同单元格的代表性图像。面板D是面板C中白色框中区域的3.5X 放大。缩放条形图: 5 µm 用于面板-c和E F;面板D的1µm。面板A中的图像与图 3A中使用的图像相同, 但包括 F 肌动蛋白通道的叠加。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/57028fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57028/57028fig5.jpg"> 图 5: 由于未表达或 GFP 融合蛋白表达不足而导致质量较差的 STED 图像的示例.A20 表达 CLIP-170-GFP 的细胞被允许在抗 IgG 涂层的盖玻片上扩散15分钟, 然后被固定并染成图4。(A, B)CLIP-170-GFP 过度表达导致 CLIP-170-GFP (A)的大、异常聚集, 并损害 MTOC 极化对抗原接触点(B)。(C)通过增加激光功率来补偿 CLIP-170-GFP 的表达不足, 从而使 STED 图像质量较差。刻度条: 5 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/57028fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57028/57028fig6.jpg"> 图 6: 主 B 细胞中微管细胞骨架的 STED 图像.原发脾 B 细胞培养为 6 h, 5 µL BAFF 加2.5 µg/毫升 LPS, 然后允许传播15分钟的盖玻片, 已涂有抗 IgM 抗体。这些细胞被固定和染色的α蛋白抗体加上 Alexa 的568共轭二次抗体, 以可视化微管。显示一个具有代表性的图像。刻度条: 5 µm.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/57028fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Movie 1" src="/files/ftp_upload/57028/57028video1.jpg"> 影片 1: B 细胞微管网络的3D 重建.A20 细胞被允许在抗 IgG 涂层盖玻片上扩散15分钟, 然后用α蛋白抗体加上 Alexa 的488共轭二级抗体进行 immunostained。Z 切片以0.2 µm 步长捕获, 共37帧。采用 STED 显微镜成像软件进行3D 重建。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57028/Movie_1-Wang.mp4" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>

Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

我们提出了一个使用 STED 显微术的协议, 同时图像肌动蛋白结构, 微管和微管加端结合蛋白的 b 细胞已扩散到盖玻片的抗体涂层的 b 细胞受体, 一个模型的初始阶段免疫突触形成。

利用受刺激的排放损耗 (STED) 显微镜可视化肌动蛋白和微管骨架在 B 细胞免疫突触中的应用

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

visualizing-actin-microtubule-cytoskeletons-at-b-cell-immune-synapse

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

13.6K 次观看.  University of British Columbia. 我们提出了一个使用 STED 显微术的协议, 同时图像肌动蛋白结构, 微管和微管加端结合蛋白的 b 细胞已扩散到盖玻片的抗体涂层的 b 细胞受体, 一个模型的初始阶段免疫突触形成。

视频: Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

测量细菌感染小鼠和免疫反应李斯特菌</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

科研

免疫与感染

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

寄生蜂简介<em&gt;果蝇</em&gt;和Antiparasite的免疫反应的

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

使用一个调查益生素对肺炎球菌定植的影响<em&gt;体外</em&gt;粘附试验

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

免疫突触由双色时间选通受激发射损耗（STED）纳米显微可视化

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

从新型H7N9流感病毒利用杆状病毒表达系统的功能重组血凝素和神经氨酸酶蛋白的表达

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

影像性流式细胞仪对人体免疫突触的定性和定量分析

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

脂肪组织中的血管和神经纤维的共染色

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.