Hematopoietic Cell Isolation by Antibody-Free Flow Cytometry in the Zebrafish Embryo

The zebrafish is a useful model to identify genes functioning in hematopoiesis, owing to high conservation of hematopoiesis. Flow cytometry is widely used to isolate and quantitatively characterize human and mouse hematopoietic cells, often using fluorescently labeled antibodies. However, such analysis is limited in zebrafish due to lack of antibodies that recognize zebrafish hematopoietic cells. We here describe methods for isolation of hematopoietic cells by antibody-free flow cytometry in the zebrafish embryo. Hematopoietic stem cells (HSCs) are specified from a specific subset of vascular endothelial cells, the hemogenic endothelial cell (HEC), by a high level of Notch signaling. In combination with a Notch reporter line (Tp1:GFP) and a vascular specific line (fli1a:dsRed), HECs can be isolated as Tp1:GFP^high fli1a:dsRed⁺ cells at 20-22 hours post-fertilization (hpf). Zebrafish erythrocytes can be purified using 1,5-bis{[2-(dimethylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9,10-dione (DRAQ5), a DNA-staining fluorescent dye, and gata1:dsRed (erythroid marker line). DRAQ5^high dsRed⁺ cells are morphologically erythrocyte-like, hemoglobin-stained positive, and express erythropoiesis-related genes. Zebrafish neutrophils can be also isolated using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5. PHA-E^low DRAQ5^low cells have myeloperoxidase activity, are Sudan Black B-positive, and express neutrophil-related genes. These methods will help to perform genetical and functional assays for various types of hematopoietic cells in zebrafish embryos.