Weavers Helen, Franz Anna, Wood Will, Martin Paul
School of Biochemistry, Biomedical Sciences, University of Bristol; School of Cellular and Molecular Medicine, Biomedical Sciences, University of Bristol;
School of Biochemistry, Biomedical Sciences, University of Bristol.
J Vis Exp. 2018 Jun 14(136):57871. doi: 10.3791/57871.
During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle - the Drosophila pupa - offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.
在对组织损伤的快速炎症反应过程中，先天性免疫系统的细胞会迅速被招募到损伤部位。一旦到达伤口，先天性免疫细胞会执行许多基本功能，如抵抗感染、清除坏死碎片以及刺激基质沉积。为了全面了解调节这种免疫反应的各种信号事件，至关重要的是在体内实时以高时空分辨率观察多个细胞谱系的复杂行为（以及它们之间发生的相互作用）。果蝇胚胎的光学透明性和遗传易处理性使果蝇成为一个极具价值的模型，可用于实时成像和剖析炎症细胞行为的基本方面，包括发育性扩散机制、凋亡小体和/或微生物病原体的清除以及向伤口的募集。然而，最近的研究表明，在果蝇生命周期的更后期阶段——果蝇蛹期——具有许多明显的优势，包括提高RNA干扰效率、延长成像时间以及显著增加免疫细胞数量。在这里，我们描述了一种在活的果蝇蛹中以高时空分辨率对伤口修复及相关炎症反应进行成像的方案。为了跟踪再上皮化和炎症的动态过程，我们针对上皮细胞和先天性免疫细胞使用了多种特定的体内荧光标记物。我们还展示了可光转换荧光团（如Kaede）在跟踪特定免疫细胞亚群方面的有效性，以追踪它们迁移到损伤部位并从损伤部位消散的行为。
