title: Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.
abstract: This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics AS CR v.v.i., Prague, Czech Republic.
authors.forename: Ondrej
authors.identifier: http://orcid.org/0000-0001-7443-175X
authors.initials: O
authors.lastname: Svoboda
authors.affiliation: Department of Biological Sciences, California State University Chico, Chico, California, USA.
authors.forename: David L
authors.initials: DL
authors.lastname: Stachura
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics AS CR v.v.i., Prague, Czech Republic.
authors.forename: Olga
authors.initials: O
authors.lastname: Machonova
authors.affiliation: Stem Cell Program, Dana-Farber/Boston Children's, Boston, Massachusetts, USA.|Division of Hematology/Oncology, Dana-Farber/Boston Children's, Boston, Massachusetts, USA.|Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Leonard I
authors.initials: LI
authors.lastname: Zon
authors.affiliation: Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.
authors.forename: David
authors.initials: D
authors.lastname: Traver
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics AS CR v.v.i., Prague, Czech Republic.
authors.forename: Petr
authors.identifier: http://orcid.org/0000-0002-6202-7650
authors.initials: P
authors.lastname: Bartunek
mesh_terms: D000818:Animals; D002347:Carps / Q000097:blood; D018929:Cell Culture Techniques; D003470:Culture Media / Q000737:chemistry; D016207:Cytokines / Q000235:genetics*; D020869:Gene Expression Profiling; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D008967:Molecular Biology / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics; D015027:Zebrafish / Q000097:blood*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D003470:Culture Media; D016207:Cytokines; D011994:Recombinant Proteins; D029961:Zebrafish Proteins