使用IL-1β启动子驱动的红色荧光蛋白记者小鼠中性粒细胞引发的活体成像
Summary
This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.
Abstract
中性粒细胞是人体血液循环中最丰富的白细胞，并迅速募集到炎症部位。底漆是增强中性粒细胞的吞噬功能的重要事件。虽然大量研究纷纷亮相感染和损伤过程中存在的和中性粒细胞引发的重要性，意味着可视化这一进程的体内已经不可用。所提供的协议使嗜中性粒细胞在活的动物引发的动态过程的监测通过组合三种方法：1）红色荧光蛋白报告信号-作为吸2） 在体内嗜中性粒细胞标记的量度-通过荧光标记的抗淋巴细胞抗原的注射来实现6G（Ly6G）单克隆抗体（mAb）和3）活体共焦成像。几个关键步骤都参与了这一协议：恶唑酮诱导小鼠耳部皮肤炎症，动物适当镇静，抗Ly6G单抗重复注射，和prev成像过程中的焦点漂移ention。虽然有一些限制已经观察到，如连续成像时间（〜8小时）在一只小鼠的限制和异硫氰酸荧光素 – 葡聚糖从血管中的炎症状态的泄漏，该协议提供用于活体成像的基本框架底漆嗜中性粒细胞的行为和功能，可以很容易地扩展到在小鼠炎症模型其他免疫细胞的检查。
Introduction
中性粒细胞在流通中最丰富的，短命的白细胞。他们迅速招募到感染或损伤部位，在那里他们通过与含有抗菌肽和蛋白酶1粒沿着活性氧和氮中间体的发布作为专业的吞噬细胞。在他们的招聘，中性粒细胞“引”的各种试剂，包括微生物产物，趋化因子和炎症细胞因子，从而在抵达机场时明显增强吞噬细胞的功能在炎症2的网站。嗜中性粒细胞引发的机制已被广泛研究的体外 3,4;然而， 在体内的过程的动态监控一直不可能日期。
最近，活体成像已成为用于可视化和量化生物过程的细胞动力学在活生物体的重要技术。 IntraviTAL成像可通过常规的单光子激发显微术来进行（ 例如，共焦）或多光子显微镜接近5。随着时间的推移，大量的改进已在该技术实现更高的图像分辨率，改善成像深度达到，组织光损伤和增强防抖6,7下降。鉴于其独特的，以使随着时间的推移细胞迁移和互动的动态可视化能力，活体显微镜已广泛应用于免疫学8应用研究的不同领域。活体成像使免疫学家更好地理解和背景情况在活的动物模型在细胞和分子水平上既免疫应答。
在转基因的最新进展以及敲入报告小鼠已用于监测中性粒细胞的动力学行为活体动物提供了有用的工具。溶菌酶M助驱动的增强型绿色荧光蛋白敲入小鼠已被广泛使用在各种炎性过程，包括外渗，细菌感染，和无菌炎症9-15来表征嗜中性粒细胞，单核细胞和巨噬细胞的运动性。此外，表达细胞质荧光共振能量转移的生物传感器的转基因小鼠已在发炎肠16内研究嗜中性粒细胞细胞外调节的有丝分裂原激酶和蛋白激酶A的活动使用。以高特异性的鼠模型用于荧光表达在嗜中性粒细胞是追赶敲入小鼠，其产生Cre重组以及荧光蛋白质tdTomato，其本身被耦合到淋巴细胞抗原6G（Ly6G）17的表达。从这个模型Ly6G缺陷嗜中性粒细胞的可视化已经证明，这些细胞中的各种无菌或感染体内炎症上下文发挥正常功能。表达红色荧光蛋白荧光p的转基因小鼠认为包括嗜中性粒细胞，炎性单核细胞，和活化的巨噬细胞 – – interleuikin-1β（IL-1β）启动子（pIL1-DsRed的）已被用于可视化的IL-1β产生细胞的能动行为鼠标的控制下rotein基因新兴在发炎的皮肤18。
体内标记可作为跟踪的中性粒细胞的细胞和分子的行为在炎症组织的另一种方法。低剂量荧光的静脉注射标记的抗Gr-1单克隆抗体（mAb），GR-1 +嗜中性粒细胞的募集级联后已被显现在小鼠皮肤损伤感染金黄色葡萄球菌 19。 体内含有链霉抗偶联物的施用缀705纳米的量子点和生物素化抗Ly6G单抗特异性标记循环嗜中性粒细胞20。此外，这样的共轭物成neutroph的内吞IL囊泡允许在嗜中性粒细胞迁移进入间质高速囊泡运输的跟踪。 体内用抗P-选择荧光标记的抗体的糖蛋白配体-1（PSGL-1），L-选择素（CD62L），整联αM标记（CD11b的）和趋化因子（CXC母题）受体2（CXCR2）在TNFα诱发炎症模式在早期的炎症已经21阐明在发挥作用的调节机制。偏振中性粒凸出PSGL-1富含腹足上活化的血小板与CD62L本交互，导致CD11b和CXCR2，驱动中性粒细胞迁移和引发炎症受体的再分配。
IL-1β是在致敏嗜中性粒细胞22升高签名基因之一。在pIL1-DsRed的报告小鼠，红色荧光蛋白的荧光信号（ 即 ，IL-1β启动子活化）与IL-1β的mRNA表达和IL-1β蛋白质的生产正相关。<SUP> 18为了监测中性粒涂刷的过程中，一个活体显微镜方法的开发涉及在pIL1-DsRed的小鼠模型皮肤炎症的诱导恶唑酮（OX）以下的中性粒细胞的荧光缀合的抗Ly6G单抗体内标记。通过这种模式，能够研究在各种疾病和病症的动物模型中引发的中性粒细胞的行为和功能。
Protocol
Representative Results
Discussion
本研究的目的是开发一种技术用于监测中性粒涂刷的过程在活的动物，这尚未得到满足由目前可用的技术。为了实现这一目标，三既定方法被执行：1）皮肤炎症的诱导IL-1β的启动子驱动的DsRed的报告小鼠作为涂刷的量度，用低剂量的荧光缀合的抗Ly6G的嗜中性粒细胞的2） 体内标记单克隆抗体，以及3）活体共聚焦显微镜成像。这三种方法的组合使炎性皮损致敏中性粒细胞（红色荧光蛋白+…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors have no acknowledgements.
Materials
| Heparin sodium | APP Pharmaceuticals | NDC 63323-540-31 | |
| ACK lysing buffer | Lonza | 10-548E | |
| Fetal bovine serum | Sigma-Aldrich | F0926 | |
| Lipopolysaccharides | Sigma-Aldrich | L4391 | |
| Ketamine hydrochloride | Hospira | NDC 0409-2051-05 | |
| Xylazine | LLOYD Laboratory | NADA #139-236 | |
| Acepromazine | Boehringer Ingelheim | ANADA 200-361 | |
| Hair-removal cream | Church & Dwight | ||
| Acetone | Fisher Scientific | A16P4 | |
| Oxazolone | Sigma-Aldrich | E0753 | |
| Alexa Fluor 647 anti-mouse Ly6G antibody | BioLegend | 127610 | |
| U-100 insulin syringe with 28 G needle | BD | 329461 | |
| FITC-CM-Dextran, 150 Kda | Sigma-Aldrich | 74817 | |
| Butterfly infusion set (27 G needle) | BD | 387312 | |
| FACSCalibur cytometer | BD | ||
| CellQuest Pro software | BD | ||
| Confocal microscope | Olympus | FV1000 | |
| Metamorph Software | Universal Imaging |
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