A novel method to purify neutrophils enables functional analysis of zebrafish hematopoiesis

Abstract

Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.