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Culture and Transfection of Zebrafish Primary Cells.

Authors
  • Russo, Giulio1
  • Lehne, Franziska2
  • Pose Méndez, Sol M2
  • Dübel, Stefan3
  • Köster, Reinhard W4
  • Sassen, Wiebke A2
  • 1 Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology; Department of Biotechnology, Institute of Biochemistry, Biotechnology and Bioinformatics, Braunschweig University of Technology.
  • 2 Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology.
  • 3 Department of Biotechnology, Institute of Biochemistry, Biotechnology and Bioinformatics, Braunschweig University of Technology.
  • 4 Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology; [email protected]
Type
Published Article
Journal
Journal of Visualized Experiments
Publisher
MyJoVE Corporation
Publication Date
Aug 17, 2018
Issue
138
Identifiers
DOI: 10.3791/57872
PMID: 30175992
Source
Medline
Language
English
License
Unknown

Abstract

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue. In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

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