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Nat Protoc. 2016 May;11(5):1007-20. doi: 10.1038/nprot.2016.053. Epub 2016 Apr 28.

Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.

Author information

1
Department of Cell Differentiation, Institute of Molecular Genetics AS CR v.v.i., Prague, Czech Republic.
2
Department of Biological Sciences, California State University Chico, Chico, California, USA.
3
Stem Cell Program, Dana-Farber/Boston Children's, Boston, Massachusetts, USA.
4
Division of Hematology/Oncology, Dana-Farber/Boston Children's, Boston, Massachusetts, USA.
5
Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, Massachusetts, USA.
6
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.

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.

PMID:
27123951
PMCID:
PMC5560128
DOI:
10.1038/nprot.2016.053
[Indexed for MEDLINE]
Free PMC Article

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