PDF(Pubmed)
Abstract：
Cells need to migrate along gradients of chemicals (chemotaxis) in the course of development, wound healing, or immune responses. Neutrophils are prototypical migratory cells that are rapidly recruited to injured or infected tissues from the bloodstream. Their chemotaxis to these inflammatory sites involves changes in cytoskeletal dynamics in response to gradients of chemicals produced therein. Neutrophil chemotaxis has been largely studied in vitro; few assays have been developed to monitor gradient responses in complex living tissues. Here, we describe a laser-wound assay to generate focal injury in zebrafish larvae and monitor changes in behaviour and cytoskeletal dynamics. The first step is to cross adult fish and collect and rear embryos expressing a relevant fluorescent reporter (for example, Lifeact-mRuby, which labels dynamic actin) to an early larval stage. Subsequently, larvae are mounted and prepared for live imaging and wounding under a two-photon microscope. Finally, the resulting data are processed and used for cell segmentation and quantification of actin dynamics. Altogether, this assay allows the visualisation of cellular dynamics in response to acute injury at high resolution and can be combined with other manipulations, such as genetic or chemical perturbations. Key features • This protocol is designed to trigger laser wound in zebrafish larvae using two-photon intravital microscopy. • The ability to wound while imaging makes it possible to monitor the behaviour and actin changes of the cells immediately after gradient exposure. • The protocol requires a two-photon microscope for best results. Compared with one-photon laser wounding, the injury is more precise and has better tissue penetration. • The focal nature of the wounds is suitable for studies of neutrophil swarming/aggregation and can be further adapted to infectious settings.
摘要：
细胞在发育过程中需要沿着化学物质的梯度迁移（趋化性），伤口愈合，或免疫反应。嗜中性粒细胞是典型的迁移细胞,其从血流中快速募集到损伤或感染的组织。它们对这些炎症位点的趋化性涉及响应于其中产生的化学物质梯度的细胞骨架动力学的变化。中性粒细胞趋化性已在体外进行了大量研究；很少开发检测方法来监测复杂活组织中的梯度反应。这里,我们描述了一种激光伤口试验，以在斑马鱼幼虫中产生局灶性损伤，并监测行为和细胞骨架动力学的变化。第一步是穿越成年鱼，收集并移植表达相关荧光报告物的胚胎（例如，Lifeact-mRuby,标记动态肌动蛋白）到早期幼虫阶段。随后，将幼虫安装并准备在双光子显微镜下进行实时成像和受伤。最后,所得数据被处理并用于细胞分割和肌动蛋白动力学的定量。总之,该测定允许以高分辨率可视化细胞动力学以响应急性损伤，并且可以与其他操作相结合，如遗传或化学扰动。主要特征•该协议旨在使用双光子活体显微镜在斑马鱼幼虫中触发激光伤口。•成像时伤口的能力使得可以在梯度暴露后立即监测细胞的行为和肌动蛋白变化。•协议需要双光子显微镜以获得最佳结果。与单光子激光损伤相比，损伤更精确，组织穿透性更好。•伤口的病灶性质适合于嗜中性粒细胞聚集/聚集的研究,并且可以进一步适应感染环境。
