A method for the in vivo measurement of zebrafish tissue neutrophil lifespan

Neutrophil function is thought to be regulated, in large part, by limitation of lifespan by apoptosis. A number of studies suggest that circulating neutrophils have a half-life of approximately 6 hours, although contradictory evidence exists. Measuring tissue neutrophil lifespan, however, is more problematic. It is thought that tissue neutrophils survive longer, perhaps with a half-life in the order of 3-5 days, but this has never been directly measured. Zebrafish are an emerging model organism, with several advantages for the study of vertebrate immunity. In zebrafish, neutrophils constitutively assume tissue locations allowing their direct study in vivo. Using a transgenic approach, neutrophils were labelled with a photoconvertible pigment, Kaede. Photoconversion parameters were optimised and the stability of the Kaede confirmed. Individual neutrophils were photoconverted by scanning a confocal 405 nm laser specifically over each cell and their survival monitored for 48 hours, revealing an in vivo half-life for zebrafish tissue neutrophils of around 120 hours (117.7 hrs, 95% CI 95.67-157.8). Laser energy did not extend neutrophil lifespan, and we conclude that this represents a lower bound for the lifespan of a resting tissue neutrophil in the developing zebrafish larva. This is the first direct measurement of the lifespan of an in vivo tissue neutrophil.