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.
Originalsprog | Engelsk |
---|---|
Artikelnummer | e4997 |
Tidsskrift | Bio-protocol |
Vol/bind | 14 |
Udgave nummer | 11 |
Antal sider | 13 |
ISSN | 2331-8325 |
DOI | |
Status | Udgivet - 2024 |
Bibliografisk note
Funding Information:We thank Kevin O\u2019Holleran and Martin Lenz of the Cambridge Advanced Imaging Centre, for their support and assistance in this work with two-photon microscopy; fish facility staff for assistance with zebrafish husbandry. M. Sarris, A. Georgantzoglou, I. Williantarra and the relevant research were supported by a Medical Research Council Career Development Award (MR/L019523/1); Wellcome Trust (204845/Z/16/Z); Isaac Newton Trust (12.21 [a]i and 19.23 [n]), a Physiological Society research grant, and a Leverhulme Trust grant (RPG-2021-226). We also acknowledge all contributors to prior work [9,12] in which this protocol is based.
Publisher Copyright:
Copyright: © 2024 The Authors.