在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞被激活的内皮细胞相互作用对实时成像
Summary
在这里，我们报告荧光活体显微镜可视化激活的内皮细胞在活体小鼠的血管炎症和血栓形成的异型血小板中性粒细胞相互作用的实验技术。显微技术将是很有价值的研究血管疾病的分子机制和病理生理条件下测试药物制剂。
Abstract
相互作用的活化的血小板和白细胞（主要是嗜中性粒细胞），活化的内皮细胞介导的血栓形成和血管炎症。1,2期间在现场小动脉损伤的血栓形成，血小板附着的激活的内皮细胞和内皮下基质蛋白支持中性粒细胞的轧制和粘附性。3相反，小静脉的炎症条件下，嗜中性粒细胞激活的内皮细胞的粘附，可以支持循环血小板的附着和蓄积。异型血小板中性粒细胞聚集需要由特定的受体计数器受体细胞之间的相互作用的顺序过程4，它是已知的，活化的内皮细胞释放粘附分子如von Willebrand因子，由此开始在高剪切条件下，血小板的附着和蓄积。5另外，支持中性粒细胞激活内皮细胞表达选择素的滚动和粘附ð细胞间粘附分子-1（ICAM-1），分别在低剪切条件下，4血小板P-选择交互与嗜中性粒细胞通过P-选择素糖蛋白配体-1（PSGL-1），从而诱导活化的嗜中性粒细胞β2整合和坚定两种类型的细胞之间的粘附。尽管在体外实验中，全血或分离的细胞，6,7异型血小板，中性粒细胞的相互作用确定的进步，这些研究不能操纵在血管疾病的氧化应激条件。在这份报告中，用鼠标血小板和中性粒细胞的标记荧光标记的特异性抗体，我们描述了一个详细的活体显微协议来监控异型相互作用的血小板和中性粒细胞活化的内皮细胞在TNF-α诱导的炎症或激光诱导提睾肌活体小鼠微血管的损伤。
Protocol
1。活体显微镜（图1A）的制备准备灌流缓冲液（125 mM氯化钠，4.5 mM KCl中，2.5mM的CaCl 2的 ，1毫摩尔的MgCl 2，和17毫摩尔的NaHCO 3，pH为7.4）。 打开上的循环水浴中，在37℃的缓冲液，以保持温度和热控制毯用氮气曝气缓冲液（5％CO 2的平衡用氮气）。 打开显微镜系统（萨特LAMBDA DG-4高速波长变换器，BX61W工作站计算机上，奥林巴斯显微镜，MPC-200多…
Representative Results
使用详细的活体显微镜分析，异型血小板中性粒细胞上的激活的内皮细胞的相互作用是可视化的荧光标记的血小板（CD42c）或嗜中性粒细胞的标记（的Gr-1）到活体小鼠抗输注。 在一个模型中的TNF-α诱导的小静脉的炎症，最轧制的中性粒细胞稳定地粘附到内皮细胞想必与ICAM-1的相互作用激活β2整合在记录期间（3-4.5小时后注射的TNF-α， 图2A 8滚动轴承和粘附…
Discussion
在这里，我们描述了一个详细的协议，实时荧光活体显微镜可视化激活的内皮细胞在血管炎症和血栓形成的异型血小板中性粒细胞的相互作用。在此之前，类似的荧光显微镜方法研究血栓形成和血管炎症的分子机制。8,12由于异型细胞与细胞间的相互作用可能是重要的，在损伤部位的血管闭塞，这种技术将是一个有价值的工具血管疾病的细胞和分子机制的研究。实时成像技术的优点是监测?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是支持的，部分由美国国立卫生研究院（：P30 HL101302和RO1 HL109439 JC）和美国心脏协会（SDG 5270005 JC）的补助金。答：Barazia是支持的，由一个T32HL007829美国国立卫生研究院培训补助金。
Materials
| Name of Reagent/Material | Company | Catalog Number | Comments |
| NaCl | Fisher Scientific | 7647-14-5 | |
| KCl | Sigma-Aldrich | 7447-40-7 | |
| CaCl2 2H2O | Sigma-Aldrich | 10035-04-8 | |
| MgCl2 6H2O | Fisher Scientific | 7791-18-6 | |
| NaHCO3 | Fisher Scientific | 144-55-8 | |
| 0.9% NaCl Saline | Hospira | 0409-4888-10 | |
| Ketamine | Hospira | 0409-2051-05 | |
| Xylazine | Lloyd | ||
| Intramedic Tubing (PE 90) | BD Diagnostics | 427421 | |
| Intramedic Tubing (PE 10) | BD Diagnostics | 427401 | |
| Murine TNF-α | R&D Systems | 410-MT | |
| Dylight 488- labeled rat anti-mouse CD42b antibody | Emfret Analytics | X488 | |
| Alexa Fluor 647-conjugated anti-mouse Ly-6G/Ly-6C (Gr-1) Antibody | BioLegend | 108418 | |
| NESLAB EX water bath/circulator | Thermo-Scientific | ||
| Olympus BX61W microscope | Olympus | ||
| TH4-100 Power | Olympus | ||
| Lambda DG-4 | Sutter | ||
| MPC-200 multi-manipulator | Sutter | ||
| ROE-200 stage controller | Sutter | ||
| C9300 high-speed camera | Hamamatsu | ||
| Intensifier | Video Scope International | ||
| Ablation Laser | Photonic Instruments, Inc. | ||
| SlideBook 5.0 | Intelligent Imaging Innovations |
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Cite This Article
Kim, K. H., Barazia, A., Cho, J. Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice. J. Vis. Exp. (74), e50329, doi:10.3791/50329 (2013).