Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging
Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish emb...
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Published in | Developmental biology Vol. 430; no. 1; pp. 18 - 31 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
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United States
Elsevier Inc
01.10.2017
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Abstract | Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.
•In depth-protocol for primary cell culture from zebrafish embryos.•Transfection of expression constructs by electroporation.•Live and subcellular imaging of different cell types. |
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AbstractList | Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model.
•In depth-protocol for primary cell culture from zebrafish embryos.•Transfection of expression constructs by electroporation.•Live and subcellular imaging of different cell types. Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model. |
Author | Lehne, Franziska Köster, Reinhard W. Sassen, Wiebke A. Dübel, Stefan Wargenau, Sven Russo, Giulio |
Author_xml | – sequence: 1 givenname: Wiebke A. surname: Sassen fullname: Sassen, Wiebke A. organization: Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany – sequence: 2 givenname: Franziska surname: Lehne fullname: Lehne, Franziska organization: Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany – sequence: 3 givenname: Giulio surname: Russo fullname: Russo, Giulio organization: Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany – sequence: 4 givenname: Sven surname: Wargenau fullname: Wargenau, Sven organization: Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany – sequence: 5 givenname: Stefan surname: Dübel fullname: Dübel, Stefan organization: Department of Biotechnology and Bioinformatics, Braunschweig University of Technology, Braunschweig 38106, Germany – sequence: 6 givenname: Reinhard W. surname: Köster fullname: Köster, Reinhard W. email: r.koester@tu-bs.de organization: Division of Cellular and Molecular Neurobiology, Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/28802829$$D View this record in MEDLINE/PubMed |
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CitedBy_id | crossref_primary_10_1016_j_bios_2021_113417 crossref_primary_10_1016_j_jneumeth_2019_04_011 crossref_primary_10_1523_JNEUROSCI_1862_18_2019 crossref_primary_10_1016_j_jneumeth_2019_108419 crossref_primary_10_12717_DR_2018_22_3_225 crossref_primary_10_1089_zeb_2020_1935 crossref_primary_10_1007_s11626_021_00622_1 crossref_primary_10_1242_bio_036475 crossref_primary_10_1016_j_nbt_2017_12_008 crossref_primary_10_3390_medicines6020061 crossref_primary_10_1111_ceo_14017 |
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Keywords | UAS Live cell imaging Vital dyes Zebrafish CMV ER GFP dap DiOC6 ptf1a Organelle dynamics DCX VAMP1 dpf PM isl1 Primary cell culture |
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SubjectTerms | Animals Cell Shape Cells, Cultured Electroporation Embryo, Nonmammalian - cytology Embryo, Nonmammalian - metabolism Flow Cytometry Imaging, Three-Dimensional Live cell imaging Macrophages - cytology Motor Neurons - cytology Neuroglia - cytology Organelle dynamics Primary cell culture Primary Cell Culture - methods Purkinje Cells - cytology Staining and Labeling Subcellular Fractions - metabolism Transfection - methods Transgenes Vital dyes Zebrafish Zebrafish - embryology |
Title | Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging |
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