title: Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.
abstract: Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.
authors.affiliation: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115, USA.
authors.forename: Virginie
authors.initials: V
authors.lastname: Esain
authors.affiliation: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115, USA.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Cortes
authors.affiliation: Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115, USA. tnorth@bidmc.harvard.edu.|Harvard Stem Cell Institute, Cambridge, MA, 02138, USA. tnorth@bidmc.harvard.edu.
authors.forename: Trista E
authors.initials: TE
authors.lastname: North
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D006410:Hematopoiesis / Q000235:genetics / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism*
keywords: CFU-C assay; Flow cytometry; Hematopoietic stem cells; Live imaging; Zebrafish embryo