Computational quantification of fluorescent leukocyte numbers in zebrafish embryos

Fluorescent transgenes with leukocyte-restricted expression have been essential to recent studies using zebrafish models of inflammation and the innate immune response. Many of the experiments performed using these models involve quantifying changes in the number of fluorescent leukocytes. Here, we describe a tool for deriving a quantitative variable proportional to fluorescent leukocyte numbers from single-plane fluorescent digital images of whole live embryos. The parameter, called "Leukocyte units," provides reliable values linearly proportional to actual leukocyte numbers in the range 50-400 leukocytes/embryo, and its performance at higher leukocyte densities remains linear. Its usefulness is demonstrated by scoring changes in leukocyte numbers following perturbation of a characterized leukocyte specification pathway.