Repository logo
 

Visualising Neutrophil Actin Dynamics in Zebrafish in Response to Laser Wounding Using Two-Photon Microscopy.

Accepted version
Peer-reviewed

Type

Article

Change log

Authors

Williantarra, Ivanna 
Georgantzoglou, Antonios 

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.

Description

Keywords

Actin dynamics, Cell migration, Chemotaxis, Laser wounding, Neutrophil, Two-photon imaging

Journal Title

Bio Protoc

Conference Name

Journal ISSN

2331-8325
2331-8325

Volume Title

Publisher

Bio-Protocol, LLC
Sponsorship
Leverhulme Trust (RPG-2021-226)
Medical Research Council (MR/L019523/1)
Isaac Newton Trust (Minute 19.23(n))
Physiological Society (Unknown)

Version History

Now showing 1 - 2 of 2
VersionDateSummary
2024-06-19 15:51:21
Published version added
1*
2024-04-17 23:30:17
* Selected version