在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜
Summary
We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.
Abstract
有效的免疫反应是依赖于血液中的白细胞的快速动员到感染或损伤的部位。 调查白细胞迁移体内对于理解白细胞跨内皮迁移和相互作用与血管内皮的分子基础是至关重要的。一种有效的方法包括在表达荧光蛋白在感兴趣的细胞转基因小鼠活体显微镜。
在这里，我们提出了一个协议，用于成像的单核细胞和中性粒细胞在CX 3 CR1 GFP /鼠标重量静脉注射橙色染料标记的嗜中性粒细胞用倒置的共聚焦显微镜。时间推移电影收集从30分钟到几个小时的成像允许的白细胞行为在肠系膜静脉两个稳态和炎性条件下分析。我们还描述的步骤，以在本地引起血管炎症TLR2 / TLR1激动剂Pam3SK4和监控subsequeNT招聘中性粒细胞和单核细胞。
所提出的技术也可以用于监测白细胞的其他人口和调查使用等刺激或转基因小鼠的分子参与了白细胞招聘或贩运。
Introduction
嗜中性粒细胞和单核细胞是先天免疫系统，它在血液中循环连续的细胞。在损伤或感染，炎症信号诱导白细胞血细胞渗出到受损和感染的组织中，本文引发细胞免疫应答1-3。白细胞动员的快速性决定了免疫应答的积极成果。这些复杂的过程依赖于特定的分子 （如选择素，血管内皮细胞结合的趋化因子）出现在发炎的内皮细胞，帮助循环白细胞和血管内皮细胞1-3之间建立接触胶。要得到牵连白细胞分子见解招募级联，可视化细胞募集的动力学和跟踪每个小区/群体的行为是重要的。一种有效的方法包括在表达荧光蛋白在感兴趣的细胞的转基因小鼠活体显微镜。
内容“>迄今为止，使用活体显微镜几种方法被开发以图像脉管4,5，例如，耳真皮脉管或肠系膜静脉由活体共聚焦显微镜成像被用来确定的鼠的Ly6C 低的单核细胞和人巡逻行为CD14 变暗 CD16 +单核细胞上的血管的稳定状态条件下6-8腔侧，鼠标提睾脉管模型通常用于监测炎症或缺血性病症中的转基因小鼠嗜中性粒细胞之下或炎症的Ly6C 高的单核细胞的行为。在这情况下，提睾通过阴囊内注射IL1β，CCL2，TNFβ或fMLP的刺激后2-4小时，组织，然后手术形象化和活体共聚焦显微镜9-11分析。以下方案描述了一种方法来监控单核细胞和中性粒细胞的同时与任何倒置荧光共聚焦显微镜。此外，我们的方法，详细介绍了如何前映像相同的容器（稳态条件）和炎症后如何遵循白细胞募集的动力学。为此目的，我们使用在CX 3 CR1 GFP /重量鼠标，其单核细胞表达eGFP的，静脉注射一种橙色色素标记的鼠中性粒细胞。使用倒置共聚焦显微镜，它是可能的（1）来跟踪和分析稳态条件和（2）以符合两个单核细胞和中性粒细胞在同一容器中的后局部炎症招募下巡逻的Ly6C 低的单核细胞。在这里，我们创建了炎症的使用TLR2 / TLR1激动剂Pam3CSK4 12。而且，这样的成像可有助于确定是否对特定敲除小鼠或在阻断抗体6,9,13的存在下进行的，在各个步骤的白细胞募集级联感兴趣的特定分子的作用。
Protocol
Representative Results
Discussion
在这个手稿中描述的方法提供了一个一致的方法来有效地研究单核细胞和中性粒细胞行为的稳态和炎症的条件下肠系膜静脉。
制剂的关键步骤是用PBS-湿的薄纸肠的固定。如果执行得当，肠系膜血管是很好的暴露在盖玻片进行图像采集。这使得感兴趣的几个领域的选择，监测肠系膜静脉白细胞行为。
我们的方案涉及肠系膜血管的外化的实验开始前，使（1?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by EMBO (to Y.E.), Foundation Machaon (to Y.E.) and SNSF (to B.A.I.). We thank the Bioimaging Core Facility for the availability of the Nikon A1r microscope and technical assistance. We thank Mrs. Clarissa Bartley for English correction.
Materials
| 5 mL polystyrene round bottom tubes | Beckton Dickinson | 352058 | |
| 10x CFI Plan Apochromat 0.45 DT:4mm | Nikon | ||
| 20x CFI Plan Apochromat VC 0.75 DT:1mm | Nikon | ||
| Cell Tracker Orange CMRA Dye | Life Technologies | C34551 | |
| EasySep Magnet | Stem Cell Technologies | 18000 | |
| EasySep Mouse Neutrophil Enrichement kit | Stem Cell Technologies | 19762 | |
| EDTA | Sigma Aldrich | E6758 | |
| FCS | PAA | A15-042 | |
| Immersion Oil Type A | Nikon | any viscous oil | |
| Life Box Temperature Control System | Life Imaging | ||
| NaHCO3 | Sigma Aldrich | S5761 | |
| NH4Cl | Sigma Aldrich | A9434 | |
| Nikon A1R confocal microscope | Nikon | A1R | inverted microscope, motorized x/y/z stage, NIS elements software |
| PBS | Life Technologies | D8537 | |
| phenol red free DMEM/F12 | Life Technologies | 21041-025 | any phenol red free medium is suitable |
| PAM3CSK4 | Invivogen | tlrl-pms | reconstitute in PBS |
| Rat serum | Stem Cell Technologies | included in EasySep Mouse Neutrophil Enrichement kit | |
| tissue culture dish 100 | TPP | 93100 |
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