Name: Culture and Transfection of Zebrafish Primary Cells (Video) | JoVE Journal
Uploaded: 2018-08-17T13:00:00.000+00:00
Duration: 8 min 51 s
Description: Learn to culture and transfect primary cells from zebrafish embryos and adult brains for high-resolution live cell imaging. This protocol enables detailed analysis of intercellular dynamics.

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5 minutes

We thank T. Fritsch, A. Wolf-Asseburg, I. Linde and S.-M. Tokarski for excellent animal care and technical support. We are grateful to all members of the K&#246;ster lab for intense and helpful discussions. We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (KO 1949/5-1) and the Federal State of Lower Saxony, Nieders&#228;chsisches Vorab (VWZN2889).

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</ol>

The authors have nothing to disclose.

Here, we present two different protocols to culture primary cells from either 2 dpf zebrafish embryos or adult zebrafish brain.
The preparation of primary cell cultures from 2 dpf zebrafish is relatively easy to perform for anyone with experience in basic cell culture techniques. However, to obtain good and reproducible results, a sufficient number of embryos as starting material is crucial (100 is the minimum). During the raising of the embryos, all possible sources of contamination must be a...

The zebrafish (Danio rerio, D. rerio) is a popular model vertebrate for numerous fields of basic and biomedical research1. Zebrafish embryos develop rapidly ex utero, are transparent, and fit under a microscope, thus providing excellent prerequisites for studying vertebrate development in a living organism. Due to the genetic tractability of zebrafish2, many stable transgenic reporter lines with cell type-specific expression of various fluorescent markers have been established allowing for the observation of specific cell populations. The zebrafish community offers a broad variety of so-called Gal4-driver lines which carry a transgene expressing the synthetic Kal4TA4 (or the KalTA3-equivalent GalFF) gene with the Gal4-DNA-binding domain of yeast fused to viral transcriptional activation domains under the control of cell type-specific enhancers. These driver lines are crossed to effector lines which carry transgenes consisting of a defined upstream activating sequence (UAS) fused to a reporter gene. The Kal4TA4 protein binds to the UAS element, thus activating the cell type-selective expression of the reporter gene3,4. This approach allows for highly diverse combinatorial studies of almost all available enhancer and reporter elements in double-transgenic animals.
However, in-depth live imaging with focus on individual cells or their subcellular contents is limited in a whole and constantly changing embryo. To address specific cell biological questions with highest resolution, the use of cell cultures is often preferable. Some cell lines of zebrafish exist, but they are considered as heavily selected5,6,7 and their propagation is often time-consuming. Furthermore, all the available cell lines are fibroblast derived, limiting experiments using cell culture to one type of cells. Therefore, we established both an efficient and easy-to-use protocol to prepare primary cells directly from zebrafish embryos and adult zebrafish brain, together with approaches to increase the longevity of the culture and to broaden the diversity of cultivated cell types. In addition, we present a procedure to transfect embryonic primary cells with expression constructs for fluorescent organelle markers. Thus, cellular morphologies and subcellular structures can be analyzed with high spatial and temporal resolution in distinct cell types which retain their key features.

<table><tbody><tr><td>&lt;u&gt;Fish lines&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>AB (wild-type)</td><td></td><td></td><td>established by Streisinger and colleagues, available from the Zebrafish International Resource Center (ZIRC)</td></tr><tr><td>Tg(&lt;em&gt;ptf1a&lt;/em&gt;:eGFP)jh1</td><td></td><td></td><td>stable transgenic line in which the enhancer of the zebrafish gene&lt;em&gt; ptf1a &lt;/em&gt;drives expression of the fluorescent protein EGFP (Parsons &lt;em&gt;et al&lt;/em&gt;., 2007)</td></tr><tr><td>Tg(XITubb:DsRed)zf148</td><td></td><td></td><td>stable transgenic line in which the&lt;em&gt; Xenopus &lt;/em&gt;neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&amp;nbsp; (Peri and N&amp;uuml;sslein-Volhard, 2008)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Equipment&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td></td><td>model 5804 R</td></tr><tr><td>ChemiDoc MP imaging system</td><td>BioRad</td><td></td><td>model XRS+, used to acquire black-and-white images of Petri dishes containing 1 da embryos</td></tr><tr><td>confocal laser scanning microscope</td><td>Leica microsystems</td><td></td><td>model SP8, equipped with 28 &amp;deg;C temperature box and a 63X objective</td></tr><tr><td>epifluorescent microscope</td><td>Leica microsystems</td><td></td><td>model DM5500B, equipped with 28 &amp;deg;C temperature box and a 40X objective</td></tr><tr><td>Gene Pulser Xcell with capacitance extender</td><td>BioRad</td><td>1652661</td><td>electroporation device</td></tr><tr><td>Horizontal shaker</td><td>GFL</td><td></td><td>model 3011</td></tr><tr><td>incubator for cell culture (28 &amp;deg;C)</td><td>Memmert</td><td></td><td>model incubator I</td></tr><tr><td>incubator for embryos (28 &amp;deg;C)</td><td>Heraeus</td><td></td><td>type B6120</td></tr><tr><td>light microscope</td><td>Zeiss</td><td></td><td>model TELAVAL 31</td></tr><tr><td>micro pipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>sterile work bench</td><td>Bio Base</td><td></td><td>with laminar flow and UV light</td></tr><tr><td>tweezers</td><td>Dumont</td><td></td><td>Style 5, Inox</td></tr><tr><td>vertical tube rotator</td><td>Labinco B.V.</td><td></td><td>model LD-79</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Software&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>Image Lab Software</td><td>BioRad</td><td></td><td>for the ChemiDoc MP imaging system from BioRad</td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td></td><td>used for counting 1 dpf embryos by applying the Count particles-tool to the respective black-and-white images; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/. (1997-2016).</td></tr><tr><td>LAS X</td><td>Leica Microsystems</td><td></td><td>for both confocal and epifluorescent microscopes from Leica Microsystems</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plasmids&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>pCS-DCX-tdTomato</td><td>K&amp;ouml;ster Lab</td><td># 1599</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-eGFP</td><td>K&amp;ouml;ster Lab</td><td># 7</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-H2B-mseCFP</td><td>K&amp;ouml;ster Lab</td><td># 2379</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-mClover</td><td>K&amp;ouml;ster Lab</td><td># 3865</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.&lt;/em&gt;, 1994)</td></tr><tr><td>pCS-MitoTag-YFP</td><td>K&amp;ouml;ster Lab</td><td># 2199</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-ss-RFP-KDEL</td><td>K&amp;ouml;ster Lab</td><td># 4330</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al&lt;/em&gt;., 1994)</td></tr><tr><td>pCS-VAMP1-mCitrine</td><td>K&amp;ouml;ster Lab</td><td># 2291</td><td>based on the backbone pCS2+ (Rupp &lt;em&gt;et al.,&lt;/em&gt; 1994)</td></tr><tr><td>pSK-UAS:mCherry</td><td>K&amp;ouml;ster Lab</td><td># 1062</td><td>based on the pBluescript-backbone of Stratagene</td></tr><tr><td>Plasmid numbers refer to the database entries of the K&amp;ouml;ster lab. Plasmids are available upon request.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Plastic and glass ware&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>BD Falcon Cell Strainer (40 &amp;micro;m)</td><td>FALCON</td><td>REF 352340</td><td>distributed by BD Bioscience, used as &amp;ldquo;landing net&amp;rdquo; to dip deyolked embryos into ethanol and to transfer them quickly to fresh cell culture medium</td></tr><tr><td>1.5 mL reaction tubes</td><td>Sarstedt</td><td>72690550</td><td></td></tr><tr><td>24-well plate</td><td>Sarstedt</td><td>83.3922</td><td></td></tr><tr><td>50 mL falconic tube</td><td>Sarstedt</td><td>62.547.004</td><td></td></tr><tr><td>96-well plate</td><td>Sarstedt</td><td>83.3924.005</td><td></td></tr><tr><td>EasyStrainer (40 &amp;micro;m)</td><td>Greiner Bio-One</td><td>542 040</td><td>with venting slots; used to filter cells after collagenase-mediated dissociation</td></tr><tr><td>electroporation cuvette (0.4 cm)</td><td>Kisker</td><td>4905022</td><td></td></tr><tr><td>glass coverslips</td><td>Heinz Herenz Medizinalbedarf GmbH</td><td>1051201</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Fisher Scientific (Menzel Gl&amp;auml;ser)</td><td>631-0845</td><td></td></tr><tr><td>Neubauer chamber</td><td>Henneberg-Sander GmbH</td><td>9020-01</td><td></td></tr><tr><td>Pasteur pipettes (plastic; 3 mL)</td><td>A. Hartenstein</td><td>PP05</td><td></td></tr><tr><td>Petri dishes (plastic; diameter 10 cm)</td><td>Sarstedt</td><td>821473</td><td>for zebrafish embryos</td></tr><tr><td>pipette tips</td><td>Sarstedt</td><td>Blue (1000 &amp;micro;l): 70762; Yellow (200 &amp;micro;l): 70760002; White (10 &amp;micro;L): 701116</td><td></td></tr><tr><td>sterile cell culture dishes (plastic; diameter 3 cm)</td><td>TPP Techno Plastic Products AG</td><td>93040</td><td></td></tr><tr><td>sterile cell culture dishes (plastic; diameter 6 cm)</td><td>Sarstedt</td><td>72690550</td><td></td></tr><tr><td>sterile Petri dishes (plastic; diameter 10 cm)</td><td>Sarstedt</td><td>83.3902</td><td>for brain dissection</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Chemicals and Reagents&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Roth</td><td>0601.1</td><td></td></tr><tr><td>4 % paraformaldehyde in 1x PBS</td><td>Sigma-Aldrich</td><td>16005</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>calcium nitrate tetrahydrate</td><td>Sigma-Aldrich</td><td>C1396</td><td></td></tr><tr><td>ethanol p.a. 100%</td><td>Sigma-Aldrich</td><td>46139</td><td></td></tr><tr><td>goat &amp;alpha;-mouse IgG (Fc specific) FITC conjugated</td><td>Thermo Fisher Scientific</td><td>31547</td><td></td></tr><tr><td>HEPES</td><td>Roth</td><td>9105.4</td><td></td></tr><tr><td>high vacuum grease</td><td>DOW CORNING</td><td>3826-50</td><td>silicon grease used for self-made glass bottom dishes</td></tr><tr><td>magnesium sulfate heptahydrate</td><td>Merck</td><td>105886</td><td></td></tr><tr><td>methylene blue</td><td>Serva</td><td>29198.01</td><td></td></tr><tr><td>Monoclonal Anti-Tubulin, Acetylated antibody</td><td>Sigma-Aldrich</td><td>T6793</td><td></td></tr><tr><td>Aqua-Poly/Mount (mounting medium)</td><td>Polyscience</td><td>18606</td><td></td></tr><tr><td>poly-L-lysine</td><td>Biochrom</td><td>L 7240</td><td></td></tr><tr><td>potasssion chloride</td><td>Merck</td><td>104938</td><td></td></tr><tr><td>Skim milk</td><td>Roth</td><td>68514-61-4</td><td></td></tr><tr><td>Texas Red-X Phalloidin</td><td>Thermo Fisher Scientific</td><td>T7471</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>E10521</td><td>Synonym: Ethyl 3-aminobenzoate methanesulfonate</td></tr><tr><td>Triton X-100</td><td>BioRad</td><td>1610407</td><td></td></tr><tr><td>Trypan Blue</td><td>Gibco by Life Technologies</td><td>15250061</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Enzymes&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>collagenase (Type 2)</td><td>Thermo Fisher Scientific</td><td>17101015</td><td>dissolve powder in cell culture medium (8 mg/mL) and sterile-filter the solution, store aliquots at -20 &amp;deg;C</td></tr><tr><td>pronase (from Streptomyces griseus)</td><td>Roche</td><td>11459643001</td><td>distributed by Sigma-Aldrich, dissolve in 30% Danieau (10 mg/mL) and store aliquots at -20 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;u&gt;Medium and solutions for cell culture&lt;/u&gt;</td><td></td><td></td><td></td></tr><tr><td>1x PBS (Dulbecco&#039;s Phosphate Buffered Saline)</td><td>Gibco by Life Technologies</td><td>14190-169</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;-independent medium</td><td>Gibco by Life Technologies</td><td>18045054</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>filtrated bovine serum (FBS)</td><td>PAN-Biotech</td><td></td><td>individual batch</td></tr><tr><td>glutamine 100x</td><td>Gibco by Life Technologies</td><td>25030081</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>Leibovitz&#039;s L-15 medium</td><td>Gibco by Life Technologies</td><td>11415049</td><td>distributed by Thermo Fisher Scientific</td></tr><tr><td>PenStrep (10,000 U/mL)</td><td>Gibco by Life Technologies</td><td>15140148</td><td>distributed by Thermo Fisher Scientific</td></tr></tbody></table>

culture and transfection of zebrafish primary cells

Culture and Transfection of Zebrafish Primary Cells

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.

Figure 1G shows a transmitted light image of a typical culture derived from wild type embryos with striated myocytes and clusters of neuron-like cells being most abundant. To identify certain cell types more easily, a transgenic line with cell type-specific expression of a fluorescent protein can be used (Figure 1H).
Transfection of a pCS2+-based plasmid<sup class="xref...

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

culture-and-transfection-of-zebrafish-primary-cells

Research

JoVE Journal

Developmental Biology

The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolation and Characterization of Single Cells from Zebrafish Embryos

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Learn to culture and transfect primary cells from zebrafish embryos and adult brains for high-resolution live cell imaging. This protocol enables detailed analysis of intercellular dynamics.

Culture and Transfection of Zebrafish Primary Cells

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