Williantarra Ivanna, Georgantzoglou Antonios, Sarris Milka
Department of Physiology Development and Neuroscience, University of Cambridge, Cambridge, UK.
Bio Protoc. 2024 Jun 5;14(11):e4997. doi: 10.21769/BioProtoc.4997.
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.
在发育、伤口愈合或免疫反应过程中，细胞需要沿着化学物质梯度迁移（趋化作用）。中性粒细胞是典型的迁移细胞，可从血液中迅速募集到受伤或感染的组织中。它们向这些炎症部位的趋化作用涉及细胞骨架动力学的变化，以响应其中产生的化学物质梯度。中性粒细胞趋化作用在很大程度上已在体外进行研究；很少有检测方法被开发用于监测复杂活体组织中的梯度反应。在这里，我们描述了一种激光伤口检测方法，用于在斑马鱼幼虫中产生局部损伤，并监测行为和细胞骨架动力学的变化。第一步是使成年鱼杂交，收集并饲养表达相关荧光报告基因（例如，标记动态肌动蛋白的Lifeact-mRuby）的胚胎至早期幼虫阶段。随后，将幼虫固定并准备好在双光子显微镜下进行实时成像和创伤处理。最后，对所得数据进行处理，并用于细胞分割和肌动蛋白动力学的定量分析。总之，该检测方法能够以高分辨率可视化细胞对急性损伤的动态反应，并且可以与其他操作（如基因或化学扰动）相结合。关键特性 • 本方案旨在使用双光子活体显微镜在斑马鱼幼虫中引发激光伤口。 • 在成像时进行创伤处理的能力使得在梯度暴露后能够立即监测细胞的行为和肌动蛋白变化。 • 该方案需要使用双光子显微镜以获得最佳结果。与单光子激光创伤相比，损伤更精确且组织穿透性更好。 • 伤口的局部性质适用于中性粒细胞聚集/聚合的研究，并且可以进一步适用于感染情况。
