Name: isolation and characterization of single cells from zebrafish embryos
Uploaded: 2016-03-12T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

Detta protokoll kräver användning av levande, vuxna zebrafisk för att producera embryon. Embryona skördas för vävnadssamling. Det är viktigt att få godkännande från lämpliga etik prövningsnämnder för att genomföra detta experiment. 1. Skaffa Lansering i Embryon <ol><li> Dagen före experimentet, förbereda friska, vuxna zebrafisk för avel. Placera en hane och en hona på motsatta sidor av en tydlig avdelare i en avel tank. </li><li> Upprepa 1.1 för så många avelstankar s...

<ol>
	<li>Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis:+toward+the+clinic.">Single-cell analysis: toward the clinic.</a> Genome medicine. 5, (2013).</li><li>Macaulay, I. C., Voet, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+genomics:+advances+and+future+perspectives.">Single cell genomics: advances and future perspectives.</a> PLoS genetics. 10, e1004126 (2014).</li><li>Marco, 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=Bifurcation+analysis+of+single-cell+gene+expression+data+reveals+epigenetic+landscape.">Bifurcation analysis of single-cell gene expression data reveals epigenetic landscape.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 5643-5650 (2014).</li><li>Yin, H., Marshall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+single+cell+analysis.">Microfluidics for single cell analysis.</a> Current opinion in biotechnology. 23, 110-119 (2012).</li><li>Garlanda, C., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+endothelial+cells.+Specific+markers.">Heterogeneity of endothelial cells. Specific markers.</a> Arteriosclerosis, thrombosis, and vascular biology. 17, 1193-1202 (1997).</li><li>Diaz-Cano, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+heterogeneity:+mechanisms+and+bases+for+a+reliable+application+of+molecular+marker+design.">Tumor heterogeneity: mechanisms and bases for a reliable application of molecular marker design.</a> International journal of molecular sciences. 13, 1951-2011 (2012).</li><li>Guo, G., 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=Mapping+cellular+hierarchy+by+single-cell+analysis+of+the+cell+surface+repertoire.">Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire.</a> Cell stem cell. 13, 492-505 (2013).</li><li>Guo, G., 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=Resolution+of+cell+fate+decisions+revealed+by+single-cell+gene+expression+analysis+from+zygote+to+blastocyst.">Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.</a> Developmental cell. 18, 675-685 (2010).</li><li>Treutlein, B., 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=Reconstructing+lineage+hierarchies+of+the+distal+lung+epithelium+using+single-cell+RNA-seq.">Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.</a> Nature. 509, 371-375 (2014).</li><li>Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+early+development+of+zebrafish.">Genetics and early development of zebrafish.</a> Trends in genetics : TIG. 5, 283-288 (1989).</li><li>Liu, J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+the+study+of+early+cardiac+development.">Zebrafish in the study of early cardiac development.</a> Circulation research. 110, 870-874 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 203, 253-310 (1995).</li><li>Zhao, S., Fung-Leung, W. P., Bittner, A., Ngo, K., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA-Seq+and+microarray+in+transcriptome+profiling+of+activated+T+cells.">Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells.</a> PloS one. 9, e78644 (2014).</li><li>McCall, M. N., McMurray, H. R., Land, H., Almudevar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+non-detects+in+qPCR+data.">On non-detects in qPCR data.</a> Bioinformatics. 30, 2310-2316 (2014).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nature. 3, 1101-1108 (2008).</li><li>Lyons, I., 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=Myogenic+and+morphogenetic+defects+in+the+heart+tubes+of+murine+embryos+lacking+the+homeo+box+gene+Nkx2-5.">Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.</a> Genes &amp; development. 9, 1654-1666 (1995).</li><li>Chen, J. N., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+tinman+homolog+demarcates+the+heart+field+and+initiates+myocardial+differentiation.">Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation.</a> Development. 122, 3809-3816 (1996).</li><li>Zhou, Y., 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=Latent+TGF-beta+binding+protein+3+identifies+a+second+heart+field+in+zebrafish.">Latent TGF-beta binding protein 3 identifies a second heart field in zebrafish.</a> Nature. 474, 645-648 (2011).</li><li>Paffett-Lugassy, N., 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=Heart+field+origin+of+great+vessel+precursors+relies+on+nkx2.5-mediated+vasculogenesis.">Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis.</a> Nature cell biology. 15, 1362-1369 (2013).</li><li>McCurley, A. T., Callard, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+housekeeping+genes+in+zebrafish:+male-female+differences+and+effects+of+tissue+type,+developmental+stage+and+chemical+treatment.">Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment.</a> BMC molecular biology. 9, 102 (2008).</li><li>Tang, R., Dodd, A., Lai, D., McNabb, W. C., Love, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+zebrafish+(Danio+rerio)+reference+genes+for+quantitative+real-time+RT-PCR+normalization.">Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization.</a> Acta biochimica et biophysica Sinica. 39, 384-390 (2007).</li><li>Serbedzija, G. N., Chen, J. N., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+in+the+heart+field+of+zebrafish.">Regulation in the heart field of zebrafish.</a> Development. 125, 1095-1101 (1998).</li><li>de Pater, 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=Distinct+phases+of+cardiomyocyte+differentiation+regulate+growth+of+the+zebrafish+heart.">Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart.</a> Development. 136, 1633-1641 (2009).</li><li>Hami, D., Grimes, A. C., Tsai, H. J., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+development+requires+a+conserved+secondary+heart+field.">Zebrafish cardiac development requires a conserved secondary heart field.</a> Development. 138, 2389-2398 (2011).</li><li>Yelon, D., Horne, S. A., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restricted+expression+of+cardiac+myosin+genes+reveals+regulated+aspects+of+heart+tube+assembly+in+zebrafish.">Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.</a> Developmental biology. 214, 23-37 (1999).</li><li>Molina, N., 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=Stimulus-induced+modulation+of+transcriptional+bursting+in+a+single+mammalian+gene.">Stimulus-induced modulation of transcriptional bursting in a single mammalian gene.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 20563-20568 (2013).</li></ol>

Den metod som beskrivs häri använder uttryck av ett fluorescerande protein under kontroll av en celltypsspecifik promotor för att berika en population av hjärt progenitorceller från zebrafisk embryon för användning i mikroflödesassisterad enda cell capture system för bedömning expression av en underuppsättning av hjärt gener i singel celler. Förutsatt att FACS laser excitations- och emissionsförmåga är kompatibla med fluoroforen (er) för val, kan denna metod användas för något fluorescerande reporter...

De flesta av dagens studier av cell- och molekylärbiologi är baserade på medelvärden befolknings. Emellertid kan viktiga biologiska händelser maskeras av dessa traditionella populationsbaserade analyser, eftersom små populationer kan spela viktiga roller i biologiska processer och sjukdomar resultatet. Förstå genuttryck i heterogena populationer vid den enda cellnivå kan (och har) leda till relevanta biologiska och kliniska insikter 1,2. Berör embryonala utvecklingsstudier, i en större population av celler, stamceller är ofta underrepresenterade, vilket gör det svårt att upptäcka subtila förändringar i genuttryck som slutligen initiativ till beslut cell öde 3. På liknande sätt kan en enda cell typ har olika expressionsprofiler som svar på mikromiljön 4. Till exempel, bosatta endotelceller i samma organ eller i olika organ (t ex., Aorta eller njure) uppvisar signifikant heterogenitet trots att dela gemensamma MORPhological och funktionella egenskaper 5. Dessutom kan cancerceller befolka samma tumör också ha varierande molekyl profiler eller mutationer vid den enda cellnivå 6. I modellsystem har transkriptomik i enstaka celler framgångsrikt identifierat nya cellpopulationer, kännetecknad mellantillstånd som uppstår under celldifferentiering, och avslöjade differential cellulära svar på stimuli 7,8,9. Sådana insikter skulle ha maskerats i konventionella populationsbaserade studier. Zebrafisk embryon är en oerhört underutnyttjade källa stam, stamfader, och differentierande celler för att utforska frågor om en enda cell heterogenitet och molekylär reglering av cellulära identiteter under utveckling. Deras mycket stereotypa, ex vivo utveckling och enkel genetisk manipulation gör dem till en utmärkt modellsystem för detta synsätt 10,11. Närmare bestämt en stor begränsning för tolkning av en enda cell gene uttryck data är att säker identifiering av nya mellancelltillstånd under utveckling kräver mycket noggrann timing av vävnad samling 9. Detta är nödvändigt för att säkerställa att heterogenitet mellan infångade cellerna representerar heterogenitet i en vävnad vid en enda tidpunkt snarare än heterogenitet i genuttryck presenteras av åldersberoende celldifferentiering. Jämfört med möss, kan zebrafisk embryoutveckling exakt synkroniseras över ett stort antal embryon 12. Dessutom, med stora kopplingsstorlekar, zebrafisk embryon kan användas som en rik källa till stam- och stamfaderceller. Detta protokoll beskriver en metod för att isolera celler från zebrafisk embryon och fånga enskilda celler med hjälp av en kommersiellt tillgänglig integrerad mikrofluidik krets (IFC) chip och autoprep system för QRT-PCR genexpressionsanalys. Detta protokoll kan vara snabbt överföras till någon hög genomströmning multiplexering analyser inklusive helatranskriptom sekvense som tillåter mer omfattande analys av cell heterogenitet 13. Det finns också flera fördelar för traditionella genuttryck analyser. Den enda cell isolering protokoll ger hög lönsamhet efter FACS, vilket minskar andelen komprometterade celler som ingår i program nedströms. Genom att använda en IFC, kan infångade cellerna observeras direkt för att utvärdera en avskiljningsnivå och bedöma cell hälsa morfologiskt. Dessutom är detta protokoll i stort sett tillämpas på zebrafisk forskarsamhället, som endast kräver en märkt transgen fisk linje och tillgång till mikroflödescell tekniker för avskiljning. Som bevis på principen, var enskilda celler härledda från hjärt stamceller isoleras och fångas på ett IFC-chip, och sedan den relativa förekomsten av hjärtdifferentieringsmarkörer mättes med QRT-PCR. Genexpressionsanalys vid den enda cellnivå visar att hjärt progenitorceller samexistera med deras differentiating avkomma. Insikten från encelliga profilering av hjärt stamceller kan belysa heterogenitet i genuttryck mönster bland hjärt progenitorceller under ryggrads utveckling, som kan ha maskerats i traditionella populationsbaserade analyser.

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplies</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11254-20</td>
			<td>For removing the chorion from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube 2 mL</td>
			<td style="width:111px;">GeneMate</td>
			<td style="width:103px;">C-3261-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 um cell strainer</td>
			<td style="width:111px;">Biobasic</td>
			<td style="width:103px;">SP104151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm culture dish</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">351008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACS tubes topped with 35 um cell strainer</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005091</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IFC chip manufacturer&#39;s protocol</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-6117</td>
			<td>Version 100-6117 E1 was used in representative experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wide bore pastuer glass pippette</td>
			<td style="width:111px;">VWR</td>
			<td style="width:103px;">14673-010</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult wild type zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used AB line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult transgenic zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used Tg(nkx2.5:ZsYellow)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents for cell dissociation</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double distilled water</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:111px;">Instant Ocean</td>
			<td style="width:103px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">S271-3</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">P5405</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">S6014</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leibovitz&#39;s L-15</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">03-600-511</td>
			<td>Heat inactivate; any brand of FBS should be fine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 1 -TrypLE</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">12605-010</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 2 - FACSmax</td>
			<td style="width:111px;">Genlantis</td>
			<td style="width:103px;">T200100</td>
			<td>Store -20; thaw on ice; bring to room temp before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pronase (optional)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">P5147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L/D Dye - Sytox Blue</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">S34857</td>
			<td>Any live/dead stain suitable for flow cytometry will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents for IFC plate use and qRT-PCR</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene-specific probes</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>Probes will vary by experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe ef1a</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03432748_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe gata4</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03443262_g1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe nk2-5</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03074126_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe myl7</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03105700_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe vmhc</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03431136_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe isl1</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03425734_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4369016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents listed in IFC manufacturer&#39;s protocol</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 Reagent Kit</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5319</td>
			<td>Reagents for loading cells onto IFC plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ambion Single Cell-to-CTTM</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4458237</td>
			<td>Reagents for reverse transcription and pre-amplification steps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Molecular Biology Quality Water</td>
			<td style="width:111px;">Corning</td>
			<td style="width:103px;">46-000CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 IFC for PreAmp (5-10 um)</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5757</td>
			<td>IFC plate for small cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 AutoPrep machine</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5477</td>
			<td>For IFC plate use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">Z359629</td>
			<td>For counting cells and assessing cell size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For removing embryos from chorion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue culture microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For assessing single cell digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS machine</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For isolating cells of interest</td>
		</tr>
	</tbody>
</table>

isolation and characterization of single cells from zebrafish embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

Som bevis i princip var genuttryck bedöms utforska differentieringsdynamik under hjärt utveckling. I zebrafisk, hjärt progenitorer uppstår från en mesodermal population av celler som migrerar till den anteriora lateralplattan mesoderm där de smälter samman för att bilda den linjära hjärta tub. Före fusion, hjärt stamceller börjar uttrycka transkriptionsfaktor Nkx2.5 (NK2 homeobox 5), som tros vara den tidigaste specifik markör för hjärt stamceller 16,17.</su...

Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

Isolation and Characterization of Single Cells from Zebrafish Embryos

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.

Isolering och karakterisering av enskilda celler från zebrafiskembryon

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

The overall goal of this experiment is to isolate single cells from early stage zebrafish embryos for their capture in a microfluidic-based single-cell multiplex system to assess the gene expression of individual cells. This method can help answer key questions in the developmental biology field, such as what transcriptional changes do cells undergo during specification and differentiation. The main advantage of this technique is that it is an unbiased approach for revealing the heterogeneity that is masked in conventional population-based techniques.Generally, individuals new to this technique will struggle because generating the viable preparations of single cells is technically challenging but essential for downstream analysis. Once mastered, processing the whole embryo samples into single lysed cells on an integrated microfluidic chip can be completed in six hours if steps are performed properly. While attempting this procedure, it's important to remember to make sure that only single cells are captured on the microfluidic device.After breeding adult wild-type and transgenic zebrafish, collect embryos at 15-minute intervals according to facility and institution-approved standard operating procedures. At least two hours later, transfer 100 to 300 fertilized embryos from a single timepoint into a new Petri dish containing fresh egg water. And use fine forceps to manually remove the chorion from each embryo.Next, use a wide bore glass pipette to transfer the embryos into a 2-milliliter microcentrifuge tube in a minimum volume of egg water. When all of the embryos have been transferred, replace the water with 1 milliliter of ice cold egg water and submerge the tube in ice for 20 minutes. After the euthanization, use a wide bore glass pipette to remove the supernatant.And a P-1000 pipette to add fresh egg water to the embryos two times. After the second wash, replace the egg water with 1 milliliter of de-yolking buffer and use a P-1000 pipette to triturate the embryos eight to 12 times until the yolk is dissolved and only the bodies of the embryos are visible. Collect the tissue by centrifugation.Then, use the pipette to gently remove the supernatant without disturbing the tissue pellet, and re-suspend the cells in 1 milliliter of fresh egg water. After washing the embryos two more times, re-suspend the pellet in 1 milliliter of room temperature cell dissociation reagent-1. Then place the tube on its side for a 10-minute incubation at room temperature.With gentle trituration every two to three minutes to prevent clumping. At the end of the incubation, spin down the cells again and re-suspend the pellet in 1 milliliter of cell dissociation reagent-2. Incubate the cells horizontally at room temperature for another five to 15 minutes with gentle trituration every two to three minutes.Every five minutes, dilute 2 microliters of supernatant in 18 microliters of FACS buffer, and dispense the cells as a droplet onto a cell culture dish. Place a coverslip over the sample and use a tissue culture microscope to assess the digestion progression at 10 and 20x magnifications. When the preparation appears to be a mix of primarily single cells with some small and large cell clusters and a few mostly intact embryo bodies, spin down the cells and re-suspend the pellet in 1 milliliter of cold FACS buffer.Next, wet a 40-micron cell strainer with FACS buffer. And filter the cells through the strainer into a 35-millimeter cell culture dish. After spinning down the cells again, re-suspend the pellet in 100 microliters of FACS buffer, and count the number of viable cells by Trypan blue exclusion.Next, dilute the sample to 5 times 10 to the 6th cells per milliliter. And reserve 10 to 20%of the cells for the unstained control. Stain the rest of the cells with a fluorescent live/dead discrimination dye.Then wet a 35-micron cell strainer capped FACS tube with 20 microliters of FACS buffer, and add the cells to the strainer, collecting them by gravity. Using the illustrated gating strategy, sort the single live cells expressing the fluorescent marker. Verifying the gating with the double-labled cells.Sort 2, 000 to 4, 000 cells from the population of interest into 5 microliters of cold FACS buffer in a microcentrifuge tube on ice. Then transfer 1 microliter of the sorted cells into a new FACS tube with 100 microliters of FACS sorting buffer and a 1 to 1, 000 dilution of live/dead stain. And run the cell sample using the sorting gates to assess the post sort viability.Next, load 1 microliter of cells diluted in 9 microliters of FACS buffer onto a hemocytometer to determine the cell concentration and average diameter. Now select an integrated microfluidic circuit, or IFC chip, of the appropriate size for the average cell diameter of the sample, and prime the fluidics according to the manufacturer's instructions. Load the cells onto the plate according to the manufacturer's instructions, and load the plate into a compatible fluidic machine.Then run an appropriate cell loading script for the IFC chip to push the cells into the capture lanes. To confirm that the cells are lodged in the capture sites, mount the plate on a microscope equipped with a plate adapter and obtain brightfield and fluorescence images of each capture site at a 10x magnification. The cells can then be processed for downstream assessment of their target gene expression by QRT-PCR.At the 18-somite stage, fluorescence microscopy can be used to verify the ZsYellow expression of the embryos. After sorting, cells with a range of diameters are obtained. The IFC plate can be used to capture cells with a diameter of interest.For example, in this experiment, cells five to 10 micrometers in diameter. As expected, the sorted and captured cells from 18-hour post-fertilization embryos expressed the ZsYellow fluorescent marker. Here, QRT-PCR was used to assess the relative gene expression of specific genes of interest in 40 single cells from cell capture sites on an IFC plate.Since the range of cycle threshold values for the housekeeping gene was too broad for comparing gene expression between samples and many cycle threshold values exceeded 30, The values were visualized as a heat map. Comparing across samples, a substantial heterogeneity in gene expression was observed in this experiment. With cells classified as Types 1 through 5 based on their gene expression pattern.Once mastered, processing the whole embryo samples into single lysed cells on an integrated microfluidic chip can be completed in six hours if the steps are performed properly. While attempting this procedure, it's important to remember that only single cells are captured on the microfluidic chip. Following this procedure, other methods like high-throughput single cell sequencing can be performed to fully characterize the transcriptomes of single cells.After its development, this technique paved the way for researchers in the field of developmental biology for exploring cellular heterogeneity in early developing embryos. After watching this video, you should have a good understanding of how to isolate single cells from zebrafish embryos, capture single cells in a microfluidic base single-cell multiplex system and assess the gene expression of individual cells.

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Isolering och Kvantitativ Immuncytokemiska karakterisering av primära myogena celler och fibroblaster från Human skelettmuskulatur

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Isolering och karakterisering av satellitceller från råtta Head Branchiomeric Muskler

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Affinity Märkning detektion av endogena receptorer från Zebrafish embryon

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

Isolering och karakterisering av mesenkymala stromala celler från human navelsträng och Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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 ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Isolering och karakterisering av navelsträngen-derived mesenkymala stamceller från prematura och benämner spädbarn

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

Isolering, kultur, karakterisering, och differentiering av humana muskelstamceller från skelettmuskulaturen biopsi förfarande

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Beredning av små RNA-bibliotek för sekvensering från tidiga musembryon

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Generation av naiva Blastoderm Explants från Zebrafish Embryon

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Rapid Isolation of Single Cells from Mouse and Human Teeth

Snabb isolering av enstaka celler från mus och mänskliga tänder

Mouse and human teeth represent challenging organs for quick and efficient cell isolation for single-cell transcriptomic or other applications. The dental ...