Name: culture and transfection of zebrafish primary cells
Uploaded: 2018-08-17T13:00:00
Description: Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Vi tackar T. Fritsch, A. Wolf-Asseburg, I. Linde och S.-M. Tokarski för utmärkt djurvård och teknisk support. Vi är tacksamma för alla medlemmar i Köster labbet för intensiv och bra diskussioner. Vi erkänner tacksamt finansiering genom den Deutsche Forschungsgemeinschafts (KO 1949/5-1) och delstaten Niedersachsen, Niedersächsisches Vorab (VWZN2889).

Alla djur arbete beskrivs här är i enlighet med rättsliga bestämmelser (EU-direktiv 2010/63). Underhåll och hantering av fisk har godkänts av lokala myndigheter och djurens välbefinnande representant Braunschweig tekniska högskolan och lägre Sachsen statligt kontor för konsumentskydd och livsmedelssäkerhet (LAVES, Oldenburg, Tyskland; AZ. §4 (02.05) TSchB TU BS).
1. beredning av primära celler från embryon som zebrafisk
<ol><li>Beredning av 2 dagar efter befruktning (dp...

<ol>
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A., Snider, L., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+embryos+regulate+the+nuclear+localization+of+XMyoD.">Xenopus embryos regulate the nuclear localization of XMyoD.</a> Genes &amp; Development. 8, 1311-1323 (1994).</li><li>Piperno, G., Fuller, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+specific+for+an+acetylated+form+of+alpha-tubulin+recognize+the+antigen+in+cilia+and+flagella+from+a+variety+of+organisms.">Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms.</a> Journal of Cell Biology. 101 (6), 2085-2094 (1985).</li><li>Barden, J. A., Miki, M., Hambly, B. D., Dos Remedios, C. 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S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nature Biotechnology. 20, 87-90 (2002).</li><li>Sassen, W. A., Lehne, F., Russo, G., Wargenau, S., D&#252;bel, S., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+zebrafish+primary+cell+culture+for+transfection+and+live+cellular+and+subcellular+imaging.">Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.</a> Developmental Biology. 430, 18-31 (2017).</li><li>Horesh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doublecortin,+a+stabilizer+of+microtubules.">Doublecortin, a stabilizer of microtubules.</a> Human Molecular Genetics. 8, 1599-1610 (1999).</li><li>Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N. G., Palmer, A. E., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22, 1567-1572 (2004).</li><li>Distel, M., Hocking, J. C., Volkmann, K., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome+neither+persistently+leads+migration+nor+determines+the+site+of+axonogenesis+in+migrating+neurons+in+vivo.">The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo.</a> Journal of Cell Biology. 191, 875-890 (2010).</li><li>Matsuda, T., Miyawaki, A., Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+protein+dynamics+inside+cells+using+a+rationally+designed+photoconvertible+protein.">Direct measurement of protein dynamics inside cells using a rationally designed photoconvertible protein.</a> Nature Methods. 5, 339-345 (2008).</li><li>Archer, B. T., Oz&#231;elik, T., Jahn, R., Francke, U., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+and+chromosomal+localizations+of+two+human+genes+encoding+synaptobrevins+1+and+2.">Structures and chromosomal localizations of two human genes encoding synaptobrevins 1 and 2.</a> Journal of Biological Chemistry. 265, 17267-17273 (1990).</li><li>Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+the+environmental+sensitivity+of+yellow+fluorescent+protein.+Mechanism+and+applications.">Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications.</a> Journal of Biological Chemistry. 276, 29188-29194 (2001).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+monomeric+green+fluorescent+protein+derived+from+Branchiostoma+lanceolatum.">A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.</a> Nature Methods. 10, 407-409 (2013).</li><li>Campbell, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monomeric+red+fluorescent+protein.">A monomeric red fluorescent protein.</a> Procedings of the National Academy of Sciences of the United States of America. 99, 7877-7882 (2002).</li><li>Peri, F., N&#252;sslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+neuronal+degradation+by+microglia+reveals+a+role+for+v0-ATPase+a1+in+phagosomal+fusion+in+vivo.">Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo.</a> Cell. 133, 916-927 (2008).</li><li>Godinho, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. 132, 5069-5079 (2005).</li><li>Jusuf, P. 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Här presenterar vi två olika protokoll till kultur primära celler från antingen 2 dpf zebrafiskar embryon eller vuxen zebrafiskar hjärnan.
Beredning av primära cellkulturer från 2 dpf zebrafiskar är relativt lätt att utföra för alla med erfarenhet av grundläggande cell kultur tekniker. Men för att få bra och reproducerbara resultat, ett tillräckligt antal embryon som utgångsmaterial är avgörande (100 är minst). Under anskaffandet av embryon, måste alla möjliga källor till...

Zebrafiskar (Danio rerio, D. rerio) är en populär modell ryggradsdjur för många fält av grundläggande och biomedicinsk forskning1. Zebrafiskar embryon utvecklas snabbt ex utero, är transparent, och passar i Mikroskop, vilket ger utmärkta förutsättningar för att studera ryggradsdjur utveckling i en levande organism. På grund av den genetiska tractability zebrafiskar2, har många stabila transgena reporter rader med cell typspecifika uttryck för olika fluorescerande markörer fastställts möjliggör observation av specifika cellpopulationer. Zebrafiskar gemenskapen erbjuder ett brett utbud av så kallade Gal4-driver-linjer som bär en transgene som uttrycker den syntetiska Kal4TA4 (eller den KalTA3-motsvarande GalFF) genen med Gal4-DNA-bindande domänen för jästen smält till viral transkriptionell aktiveringen domäner under kontroll av cell typspecifika smakförstärkare. Dessa förare linjer korsas till effektor linjer som bär transgener som består av en definierad uppströms aktiverande sekvens (UAS) smält till en reporter gen. Kal4TA4 proteinet binder till elementet UAS, alltså aktivera cellen typ-selektiv uttrycket av reporter gen3,4. Detta tillvägagångssätt möjliggör mycket skiftande kombinatoriska studier av nästan alla tillgängliga enhancer och reporter element i dubbel-transgena djur.
Djupgående levande imaging med fokus på enskilda celler eller subcellulär innehållet begränsas dock i ett hela och ständigt föränderliga embryo. För att lösa specifika cellen biologiska frågor med högsta upplösning, är användningen av cellkulturer ofta att föredra. Vissa cellinjer av zebrafisk finns, men de är ansedda som kraftigt markerade5,6,7 och deras förökning är ofta tidskrävande. Dessutom cellinjerna alla de tillgängliga fibroblast härrör, begränsa experiment med cellkultur till en typ av celler. Därför etablerat vi både ett effektivt och lätt-till-använda protokoll för att förbereda primära celler direkt från zebrafisk embryon och vuxen zebrafiskar hjärnan, tillsammans med metoder att öka livslängden på kulturen och att bredda mångfalden av odlade celltyper. Dessutom presenterar vi ett förfarande för att transfect embryonala primära celler med uttrycket konstruktioner för fluorescerande organell markörer. Således, cellulära morfologier och subcellulära strukturer kan analyseras med hög rumsliga och temporal upplösning i olika celltyper som behåller sin viktiga funktioner.

<table>
	<tbody>
		<tr>
			<td>Fish lines</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(ptf1a:eGFP)jh1</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the enhancer of the zebrafish gene ptf1a drives expression of the fluorescent protein EGFP (Parsons et al., 2007)</td>
		</tr>
		<tr>
			<td>Tg(XITubb:DsRed)zf148</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which the Xenopus neural-specific beta tubulin promoter drives expression of the fluorescent protein DsRed&#160; (Peri and N&#252;sslein-Volhard, 2008)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</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 &#176;C temperature box and a 63 x objective</td>
		</tr>
		<tr>
			<td>epifluorescent microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>model DM5500B, equipped with 28 &#176;C temperature box and a 40 x 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 &#176;C)</td>
			<td>Memmert</td>
			<td></td>
			<td>model incubator I</td>
		</tr>
		<tr>
			<td>incubator for embryos (28 &#176;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pCS-DCX-tdTomato</td>
			<td>K&#246;ster Lab</td>
			<td># 1599</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-eGFP</td>
			<td>K&#246;ster Lab</td>
			<td># 7</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-H2B-mseCFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2379</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-mClover</td>
			<td>K&#246;ster Lab</td>
			<td># 3865</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-MitoTag-YFP</td>
			<td>K&#246;ster Lab</td>
			<td># 2199</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-ss-RFP-KDEL</td>
			<td>K&#246;ster Lab</td>
			<td># 4330</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pCS-VAMP1-mCitrine</td>
			<td>K&#246;ster Lab</td>
			<td># 2291</td>
			<td>based on the backbone pCS2+ (Rupp et al., 1994)</td>
		</tr>
		<tr>
			<td>pSK-UAS:mCherry</td>
			<td>K&#246;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&#246;ster lab. Plasmids are available upon request.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plastic and glass ware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Cell Strainer (40 &#181;m)</td>
			<td>FALCON</td>
			<td>REF 352340</td>
			<td>distributed by BD Bioscience, used as &#8220;landing net&#8221; 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 &#181;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&#228;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 &#181;l): 70762; Yellow (200 &#181;l): 70760002; White (10 &#181;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals and Reagents</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 1 x PBS</td>
			<td>Sigma-Aldrich</td>
			<td>16005</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,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 &#945;-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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</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 &#176;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 &#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Medium and solutions for cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 x PBS (Dulbecco&#39;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>CO2-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 100 x</td>
			<td>Gibco by Life Technologies</td>
			<td>25030081</td>
			<td>distributed by Thermo Fisher Scientific</td>
		</tr>
		<tr>
			<td>Leibovitz&#39;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 units/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

Zebrafiskar embryon är transparenta och utvecklas utanför mamman, vilket möjliggör utmärkta i vivo imaging av dynamiska biologiska processer i en intakt och utveckla ryggradsdjur. Den detaljerad avbildning av morfologier av olika celltyper och subcellulära strukturer är dock begränsad i hela fästen. Därför etablerade vi en effektiv och lätt-till-använda protokollet till kultur levande primära celler från zebrafisk embryon och adult vävnad.
I korthet är 2 dpf zebrafiskar embryon dechorionated, deyolked, steriliseras och dissocierade att enstaka celler med kollagenas. Efter en filtrering steg är primära celler pläterade på glas botten rätter och odlat i flera dagar. Färsk kulturer, som lång sikt differentierade sådana, kan användas för högupplösta confocal imaging studier. Kultur innehåller olika celltyper, med tvärstrimmig myocyter och nervceller att vara framträdande på poly-L-lysin beläggning. Specifikt etikett subcellulära strukturer av fluorescerande markör proteiner etablerat vi också en elektroporation protokoll som möjliggör transfection av plasmid DNA till olika celltyper, däribland nervceller. Således, i närvaro av operatören definierade stimuli, komplexa cell beteende och intracellulära dynamics av primära zebrafiskar celler kan bedömas med hög rumsliga och temporal upplösning. Dessutom, med hjälp av vuxen zebrafiskar hjärnan, visar vi att tekniken som beskrivs dissociation, liksom culturing grundförutsättningarna, också arbeta för vuxna zebrafiskar vävnad.

Figur 1 g visar en överförda ljus bild av en typisk kultur från vildtyp embryon med tvärstrimmig myocyter och kluster av neuron-liknande celler är mest förekommande. För att lättare identifiera vissa celltyper, en transgena linje med cell typspecifika uttryck av en fluorescerande protein kan vara används (figur 1 H).
Transfection en pCS2 +-baserat plasmid<sup c...

Watch this Scientific Journal Video about Culture and Transfection of Zebrafish Primary Cells at JoVE.com

Culture and Transfection of Zebrafish Primary Cells

Vi presenterar ett effektivt och lätt-till-använda protokoll för att förbereda primära cellkulturer av zebrafisk embryon för transfection och levande cell imaging samt ett protokoll att förbereda primära celler från vuxna zebrafiskar hjärna.

Kultur och Transfection zebrafiskar primära celler

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

Research

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Isolering och karakterisering av enskilda celler från zebrafiskembryon

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Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

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Single-cell Photoconversion in Living Intact Zebrafish

Single-cell Photoconversion i levande intakt zebrafiskar

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

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