Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging

• Published in: Developmental biology Vol. 430; no. 1; pp. 18 - 31
• Main Authors: Sassen, Wiebke A., Lehne, Franziska, Russo, Giulio, Wargenau, Sven, Dübel, Stefan, Köster, Reinhard W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2017
• Subjects: 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; UAS; Live cell imaging; Vital dyes; Zebrafish; CMV; ER; GFP; dap; DiOC6; ptf1a; Organelle dynamics; DCX; VAMP1; dpf; PM; isl1; Primary cell culture
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2017.07.014

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.