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...

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><tbody><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</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>&amp;nbsp;R0883</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>12483020</td><td>Heat inactivate at 56 &lt;sup&gt;o&lt;/sup&gt;C 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>&lt;strong&gt;Additional materials for primary B cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70-&amp;micro;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 &lt;em&gt;E. coli &lt;/em&gt;0111:B4</td></tr><tr><td>B cell-activating factor (BAFF)</td><td>R&amp;amp;D Systems</td><td>2106-BF-010</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Transfection&lt;/strong&gt;</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 &lt;em&gt;et al.&lt;/em&gt; &lt;strong&gt;Cell&lt;/strong&gt; 109:873-885, 2002</td></tr><tr><td>&lt;strong&gt;Recommended transfection reagents and equipment &lt;/strong&gt;</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&#039;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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Coating coverslips&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Staining&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Millipore Sigma</td><td>C1016</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S374-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Microscopy&lt;/strong&gt;</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?...

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

Research

JoVE Journal

Immunology and Infection

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

13.7K 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

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 ...

科研

免疫与感染

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 ...

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 ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

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 ...

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 ...

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.

研究免疫突触形成过程中B细胞中的细胞动力学

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

成像人类免疫学突触

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Source: Zheng, P., et al. <a href="https://app.jove.com/v/52502">Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer.</a> J. Vis. Exp. (2015).
This video demonstrates a technique for visualizing the formation of the immunological synapse in natural killer (NK) cells on a supported lipid bilayer (SLB). The SLB is first labeled with a fluorophore-tagged NK cell activating receptor ligand, followed by incubation with NK cells. Subsequently, the cells are stained for polymerized actin and perforin-positive lytic granules. This approach enables the visualization of synapse formation using stimulated emission depletion (STED) microscopy.

在支撑的脂质双层上 NK 细胞免疫突触形成的超分辨率成像

1. Preparation of Liposomes

	Calculate the amount of chloroform-suspended stock solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

Assessing Immunological Synapse Topology through Live-Cell Imaging

Source: Martinelli, R. et al., <a href="https://app.jove.com/v/53288">An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates live-cell imaging of immunological synapse topology, investigating the interactions between T lymphocytes and epithelial cells carrying fluorescent antigens. Fluorescence microscopy reveals red cytoplasm displacement and yellow membrane rings in endothelial cells, indicating the formation of immunological synapses.

通过活细胞成像评估免疫突触拓扑

1. Preparing Human CD4+&#160;Th1 Effector/Memory T Cells

	Apply a tourniquet to the arm of the donor, wipe the vein with alcohol, and insert a needle. ...

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

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