双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用
Summary
在本研究中, 我们提出了一个协议, 执行双光子活体成像和细胞相互作用分析的小鼠气管粘膜感染后流感病毒。这个协议将是相关的研究人员研究免疫细胞动力学在呼吸道感染。
Abstract
细胞和细胞-病原体相互作用的分析是了解感染免疫反应动力学的重要工具。双光子活体显微镜 (2 p-病媒) 允许观察活体动物深层组织中的细胞相互作用, 同时尽量减少在图像采集过程中产生的漂白。迄今为止, 对2种对淋巴和非淋巴器官的 p 病媒进行了不同的模型描述。然而, 由于与动物呼吸循环有关的运动, 呼吸器官的成像仍然是一个挑战。
在这里, 我们描述了一个协议, 以可视化在体内免疫细胞相互作用的小鼠的气管感染流感病毒使用 2 p-病媒。为此, 我们开发了一种自定义成像平台, 包括气管的手术暴露和插管, 其次是获取粘膜上皮中中性粒细胞和树突状细胞 (DC) 的动态图像。此外, 我们详细的步骤, 以执行流感鼻腔感染和流动细胞分析的免疫细胞在气管。最后, 分析了中性粒细胞和直流运动及其在电影过程中的相互作用。该协议允许生成稳定和明亮的4D 图像, 用于评估气管内细胞间的相互作用。
Introduction
双光子活体显微术 (2 p-病媒) 是一种有效的技术, 实时成像细胞间的相互作用, 因为它们发生在他们的自然环境1。这种方法的一个主要优点是, 它允许在更大的试样深度 (500 µm 到1毫米) 的细胞过程研究与其他传统成像技术2相比。同时, 两光子激光器产生的两个低能光子的使用最大限度地减少了通常与图像采集过程2相关的组织光损伤。在过去的十年中, 2 p-病媒应用于研究不同类型的细胞相互作用的几个学科3,4,5。这些研究特别相关的免疫细胞, 其特点是其高活力和形成明显的接触后, 其他细胞和环境产生的信号。2对病原体与宿主6之间的相互作用也进行了研究。事实上, 以前已经表明, 一些病原体可以改变免疫细胞之间接触的类型和持续时间, 从而妨碍免疫应答7。
气道粘膜是第一个场所, 其中免疫反应的空气病原体产生8。因此,在体内分析这种组织中的病原宿主相互作用, 对于了解宿主防御机制在感染过程中的启动至关重要。然而, 2 的气道病媒是挑战, 主要是由于动物的呼吸周期产生的文物, 这破坏了图像采集过程。最近, 不同的手术模式被描述为成像小鼠气管9,10,11,12和肺部13,14,15, 16。气管 2 p-病原学模型代表了一个良好的设置, 以可视化的初期阶段的免疫反应在上呼吸道, 而肺肺泡 2 p-病原细胞模型更适合研究晚期感染。肺模型的存在与充气肺泡的存在有关, 限制了激光的光穿透, 使肺内呼吸道的黏膜层无法在体内成像17.反之, 由连续上皮形成的气管结构有利于图像的采集。
在这里, 我们提出了一个协议, 其中包括详细说明了执行流感感染所需的步骤, 动物的外科准备, 和2对气管的 p 病媒。此外, 我们描述了一个具体的实验设置, 以可视化中性粒细胞和树突状干细胞 (DC), 两种免疫细胞类型, 发挥重要作用的调解机制, 以抵御流感病毒18,19.最后, 我们描述了一种分析中性粒细胞-DC 相互作用的过程。这些接触已经显示, 以调节 DC 激活, 并随后, 影响免疫反应的病原体20。
Protocol
所有涉及老鼠的动物程序都是按照瑞士联邦兽医局的指导方针和动物协议进行的, 当地兽医当局已经批准。 1. CD11c-YFP 小鼠的流感感染 安全性注: Rico/8/34 甲型h1n1 流感 (PR8) 的老鼠适应菌株生长在受精卵, 纯化和滴定如前所述21。根据生物安全水平 (BSL) 2 条件, 在生物安全柜下进行了涉及受感染动物或生物样品的所有步骤。 在感染程…
Representative Results
在这项工作中, 我们描述了一个详细的协议来研究体内的运动和中性粒细胞和 DC 之间的相互作用, 在流感感染的小鼠气管 (图 3A)。为此, 我们分离 CFP+中性粒细胞 (92% 纯度;图 3B)从 CK6-ECFP 鼠, 我们过继转输转移到 CD11c-YFP 鼠感染流感。在那之后, 我们在3天的私家侦探身上执行了2的气管内病媒。此时, 我们观察到感染?…
Discussion
这项工作提供了一个详细的协议, 以生成4D 图像显示过继转输转移中性粒细胞的迁移及其与 DC 的相互作用, 在流感感染的小鼠气管。描述的 2 p-病媒模型将是相关的研究免疫细胞动力学在呼吸道感染。
最近, 在气道细胞动力学的可视化的几个模型被开发了9,10,11,12,<sup class="x…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了瑞士国家基金会 (SNF) 赠款 (176124、145038和 148183)、欧洲委员会居里夫人重返社会补助金 (612742) 和 SystemsX.ch D.U.P. (2013/124) 赠款的支持。
Materials
| Gigasept instru AF | Schülke & Mayr GmbH | 4% solution | |
| CD11c-YFP mice | Jackson Laboratories | 008829 | mice were bred in-house |
| CK6-ECFP mice | Jackson Laboratories | 004218 | mice were bred in-house |
| 1 X Dulbecco's Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride | Sigma | D8537-500ML | |
| 10 X Dulbecco's Phosphate Buffered Saline modified without Calcium Choride and Magnesium Chloride | Sigma | D1408-500ML | |
| Percoll PLUS | Sigma | E0414-1L | Store at 4°C |
| Ketamin Labatec | Labatec Pharma | 7680632310024 | Store at RT, store at 4°C when in solution of ket/xyl mixture |
| Rompun 2% (Xylazin) | Bayer | 6293841.00.00 | Store at RT, store at 4°C when in solution of ket/xyl mixture |
| 26 G 1 mL Sub-Q BD Plastipak | BD Plastipak | 305501 | |
| 30 G 0,3 mL BD Micro-Fine Insulin Syringes | BD | 324826 | |
| Falcon 40 µm Cell Strainer | Corning | 352340 | |
| 2 mL Syringes | BD Plastipak | 300185 | |
| Microlance 3 18 G needles | BD | 304622 | |
| Introcan Safety 20G (catheter) | Braun | 4251652.01 | |
| 6 Well Cell Culture Cluster | Costar | 3516 | |
| RPMI medium 1640 + HEPES (1X) | ThermoFisher Scientific | 42401-018 | Store at 4°C |
| Liberase TL Research Grade | Roche | 5401020001 | Store at -20°C / collagenase (I and II) mixture |
| DNAse I | Amresco (VWR) | 0649-50KU | Store at -20°C |
| CellTrace Violet stain | ThermoFisher Scientific | C34557 | Store at -20°C |
| EDTA | Sigma | EDS-500G | |
| Fetal Bovine Serum | Gibco | 10270-106 | Store at -20°C |
| PE-10 Micro Medical Tubing | 2Biological Instruments SNC | #BB31695-PE/1 | |
| Surgical Plastic Tape | M Plast | ||
| Viscotears | Bausch & Lomb | Store at RT | |
| Plasticine | Ohropax | ||
| High Tolerance Glass Coverslip 15mm Round | Warner Instruments | 64-0733 | |
| SomnoSuite Portable Animal Anesthesia System | Kent Scientific | SS-01 | |
| Nuvo Lite mark 5 | GCE medline | 14111211 | |
| MiniTag (gaseous anesthesia and heating bench) | Tem Sega | ||
| SURGICAL BOARD | University of Bern | ||
| TrimScope II Two-photon microscope | LaVision Biotec | ||
| Chameleon Vision Ti:Sa lasers | Coherent Inc. | ||
| 25X NA 1.05 water immersion objective | Olympus | XLPLN25XWMP2 | |
| The Cube&The Box incubation chamber and temperature controller | Life imaging Services | ||
| Imaris 9.1.0 | Bitplane | Imaging software | |
| GraphPad Prism 7 | GraphPad | Statistical software |
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Citar este artigo
Palomino-Segura, M., Virgilio, T., Morone, D., Pizzagalli, D. U., Gonzalez, S. F. Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy. J. Vis. Exp. (138), e58355, doi:10.3791/58355 (2018).