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

Katsuhiro Konno, Jingjing Kobayashi-Sun, Fumio Arai, Isao Kobayashi, Daisuke Sugiyama

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

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:GFPhigh 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). DRAQ5high 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-Elow DRAQ5low 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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages171-180
Number of pages10
DOIs
Publication statusPublished - 2022
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume2520
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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