Wir möchten Dan Carlin für Hilfe bei der Herstellung Käfig Fluorescein. Darüber hinaus möchten wir die folgenden Finanzierungsquellen danken: Vanderbilt University Medical School Program für Entwicklungsbiologie Training Grant zu JAC und NIH HD054534to JTG.

1. Synthese von Fluorescein Dextran Caged <ol><li> Messen Sie 3,5 bis 4 mg Aminodextran und fügen in die Invitrogen bereitgestellte getönte Röhre mit 1 mg CMNB-Käfig Fluorescein SE. In unseren Händen dieses Verhältnis ergibt eine durchschnittliche Belastung von ~ 2,5 Farbstoffmoleküle pro Dextran. </li><li> Add 500 ul von 0,1 M Na 2 B 4 O 7 (Natriumborat) Puffer, um das Rohr. </li><li> Cap und Vortex für 30 Sekunden, um die Aminodextran und eingesperrt Fluorescein aufzulösen. </li><li> Lassen Sie reagieren über Nacht auf einem Vortex-Mischer. </li><li> Twist off unten Schließung auf einer Zeba Spin-Säule und lösen Sie die Kappe. Markieren Sie einen Punkt auf der Säule mit einem Filzstift. Legen Spalte in einer 15-mL konischen Rohr. </li><li> Zentrifugation bei 1000 xg für 2 min mit dem Punkt vor der Mitte des Rotors. Verwenden Sie die Spalte sofort nach dem Verdichten. </li><li> Ort verdichtet Spalte in eine neue 15-mL konischen Rohr. </li><li> Pool Reaktionsgemisch und Transfer zum Zentrum des verdichteten Spalte Harzbett. Wieder aufsetzen. </li><li> Zentrifugation bei 1000 xg für 2 min mit dem Punkt vor der Mitte des Rotors. Nach dem Spin, wird dem Eluenten und der Kopf der Kolonne in etwa die gleichen Schatten in der Farbe gelb. </li><li> Übertragen Sie die gelbe Eluent (~ 350 mL) zu einer 1,5-ml-Reaktionsgefäß. </li><li> Lyophilisieren zur Trockne Speedvac. Decken Sie die Speedvac mit Folie. </li><li> Re-suspend die daraus resultierenden ~ 2-3 mg hellgelber Schaum im Wasser bis zu einer Endkonzentration von 1% w / v. </li><li> Bewahren Sie diese Lösung bei -20 ° C in Folie abgedeckten Rohre. Es ist eine gute Idee, Aliquot der Dextranlösung, um sich wiederholende Einfrieren und Auftauen zu vermeiden. </li></ol> 2. Die Injektion von Caged Fluorescein Dextran In Zebrafisch-Embryos <ol><li> Verdünnen Sie 1% w / v Bestand Käfig Fluorescein Dextran 1:5 oder 1:10 in 0,2 M KCl. </li><li> Inject 0,5 - 1.0nL der Käfig Fluorescein-Lösung in den Dotter von einer Zelle Embryonen mit einem Injektor. Nach der Injektion muss Kupplungen von Embryonen im Dunkeln bei 28,5 ° C gehalten werden, um unspezifische Uncaging reduzieren. </li><li> Entfernen Sie manuell Chorion aus Embryonen. </li><li> Anesthetize Embryonen in 4% Tricaine. </li><li> Embryonen werden in 0,8% Agarose in 35mmx10mm Petrischale angebracht werden, dann mit Ei mit Wasser bedeckt. </li><li> Äquilibrieren geschmolzener Agarose in 50 ° C Hitze-Block für mindestens 15 Minuten. </li><li> Mit einer Glaspipette, Ausarbeitung eines Embryos in einer minimalen Menge Wasser und geben es in die Agarose. Hinweis: Es ist wichtig, um das Rohr von Agarose in Ihrer Hand kühl für ca. 30 Sekunden vor der Inbetriebnahme des Embryos in die Röhre zu vermeiden Tötung des Embryos. </li><li> Pipette der Embryo aus der Tube, zusammen mit etwa 50 ul Agarose und werfen den Inhalt auf der Mitte der Petrischale. </li><li> Schnell orientieren des Embryos mit einem Kunststoff-Faden mit den Zellen zu uncaged nach oben zeigt. Hinweis: Die Agarose mit kühlen schnell so voreilig sein mit der Ausrichtung des Embryos. Embryonen können beschädigt werden, wenn manipuliert, nachdem die Agarose erstarrt sein. </li><li> Lassen Sie die Agarose vollständig erstarren (das dauert ein paar Minuten) und füllen die Schüssel zu zwei Dritteln gefüllt mit Ei Wasser mit 4% Tricaine und 0,003% PTU. </li></ol> 3. Laser Uncaging von Fluorescein Dextran <ol><li> Das Mikroskop für Uncaging verwendet muss einen 40X Wasserimmersionsobjektiv. </li><li> Wir verwenden eine Photonics Instruments Laser mit der 365 nm Farbstoff-Zelle und die 70% / 30% aufgeteilt dichroitischen Spiegel. </li><li> Vor Uncaging, muss der Laser gemacht parfokal mit dem Ziel und abgeschwächt auf die entsprechende Leistung sein. </li><li> Zur Fokussierung des Lasers, statt eines gespiegelten Objektträger unter das Ziel und Schwerpunkt der Ziel bis Kratzer in der Folie sichtbar sind. </li><li> Passen Laser Abschwächer bis der Laser eine deutlich sichtbare Kratzer in den Spiegel mit einem einzigen Impuls erzeugen können. </li><li> Passen Sie das Ziel konzentrieren rauf und runter. Wenn der Laser einen Kratzer produzieren kann, wenn das Ziel ist unscharf, passen Sie die Laserfokus ¼ Drehung nach oben oder unten. Wiederholen, bis der Laser die Spiegel nur, wenn das Ziel fokussiert ist verkratzen können. </li><li> Ort montiert Embryo unter dem Objektiv und Fokussierung auf den Bereich um uncaged werden. </li><li> Um Uncage, auf eine Zelle von Interesse konzentrieren und Puls ist 10-20 mal bei 2-3 Hz. </li><li> Nach Uncaging, kostenlos den Embryo durch Schälen Agarose entfernt mit einer Pinzette und setzen sie in Ei-Wasser mit 0,003% PTU. Lassen Embryonen in einer lichtdichten Box zu entwickeln, bis sie fixiert sind. </li></ol> 4. Antikörper Markierung und Detektion von Uncaged Fluorescein Dextran <ol><li> Fix Embryonen in Ab Fixativ (4% Paraformaldehyd, 0,3 CaCl 2, 8% Saccharose, Buffered 1X Phosphat Saline, pH 7,3) für zwei Minuten vor vier Stunden bei Raumtemperatur (RT) oder über Nacht (O / N) bei 4 ° C. Keep Embryonen in einem lichtdichten Kasten oder Folie eingeschweißt Rohr für die Etikettierung Schritte. </li><li> Entfernen Fixiermittel und ersetzen mit 100% MeOH, lassen für 10 Minuten bei RT, entfernen MeOH und Replace mit frischen 100% MeOH. </li><li> Shop in MeOH bei -20 ° C für mindestens 30 Minuten. Hinweis: Sie können in MeOH für bis zu zwei Wochen lagern, bevor Epitope zu beeinträchtigen beginnen. </li><li> Rehydrieren Embryonen mit 5 Minuten Wäschen bei RT von 75% MeOH/1X Phosphat-gepufferte Kochsalzlösung mit 0,1% Tween 20 (1X PBSTw), dann 50% MeOH/50% 1XPBTw, dann 25% MeOH/75% 1X PBSTw. </li><li> Dreimal für 5 Minuten in 1XPBSTw. </li><li> Wenn Embryonen&gt; 24 Stunden alt sind, permiabilize Embryonen mit 10 pg / ml Proteinase K in 1XPBSTw. Permiabilization ist abhängig von der Bühne. Fünf Minuten in Proteinase K ist ausreichend, wenn die Embryonen zwischen 24 und 48 Stunden nach der Befruchtung (HPF) sind. Mehr Zeit kann, wenn für ältere Larven erforderlich. Re-fix in Ab Fixativ für 20 Minuten bei RT </li><li> Wash fünf Mal für 5 Minuten in 1XPBSTw bei RT. </li><li> Inkubieren in Block-Lösung (1XPBS, 0,1% Triton x100, 10% Schafserum, 10% Rinderserumalbumin, 1% Dimethylsulfoxid) für 1-2 Stunden bei RT. </li><li> Verdünnen primären Antikörper (Anti-Fluorescein sowie Antikörper gegen Zelltyp spezifische Marker) in Blocking-Lösung. </li><li> Inkubieren Embryonen O / N bei 4 ° C in Lösung mit primären Antikörpern. </li><li> Wash viermal für 20 Minuten in 1XPhosphate Kochsalzlösung mit 0,1% Triton x100 (PBSTx) bei RT. </li><li> Verdünnen fluoreszenzmarkierten Sekundärantikörper (in der Regel 1:300) in Blocking-Lösung. </li><li> Inkubieren O / N bei 4 ° C in Lösung mit Sekundärantikörper. </li><li> Wash viermal für 20 Minuten in 1XPBSTx bei RT. </li><li> Klare Embryonen in 50% glycerol/50% 1XPBS für mindestens zwei Stunden bei RT, dann entfernen Glycerin / PBS-Mischung und 100% Glycerin und stehen lassen O / N bei 4 ° C. </li></ol> 5. Repräsentative Ergebnisse: Nach Immunomarkierung, sollte es eine angereicherte Bereich der Färbung in den Zellen, die uncaged (Figure.1) waren. Es ist nicht ungewöhnlich, dass ein relativ hohes Signal-Rausch-Verhältnis sehen in&gt; 48 Stunden alten Zebrafisch, durch spontane Uncaging der Fluorescein. <img src="/files/ftp_upload/2672/2672fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Lineage Kennzeichnung von Zebrafisch Zirbeldrüse komplex. Bei 24 HPF, einen Teil der vorderen Epiphyse anlage, durch foxd3 angegeben:. GFP-Expression, uncaged wurde mit UV-Laserpulsen A) Durch 48hpf, rief einer linksseitigen Zellklumpen die parapineal (roter Kreis) hat von der uncaged entstanden Domain (weißer Kreis). B) Uncaged Fluorescein-Signal wird in der parapineal (roter Kreis angereichert) und das Pinealorgan (weißer Kreis).

<ol>
	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., 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=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

Wie beschrieben, stellt dieses Protokoll eine relativ schnelle Linie Kennzeichnung Methode in Zebrafisch, dass bei den üblicherweise verwendeten Techniken der Mikroinjektion, Mikroskopie und Immunfluoreszenz gebaut wird. Wir haben festgestellt, dass Laser auf die effizienteste und kostengünstigste Weg, um Fluorescein in einer lokalisierten Mode Uncage werden Uncaging. Diese Methode könnte auf Linie Etikett mit experimentellen Endpunkte werden so spät wie 4 dpf verwendet. Doch wie sich Zellen teilen, die caged-Fluore...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CMNB-caged fluorescein SE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C20050</td>
<td>&nbsp;</td>
<tr>
<td>Aminodextran, 10kDa</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1860</td>
<td>&nbsp;</td>
<tr>
<td>Zeba desalt spin columns</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>89889</td>
<td>&nbsp;</td>
<tr>
<td>goat anti-fluorescein antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11095</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 633 Donkey anti-goat antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21082</td>
<td>&nbsp;</td>
<tr>
<td>35mmx10mm petri dishes</td>
<td>&nbsp;</td>
<td>Becton-Dickinson</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Upright compound microscope with 40x water immersion objective</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DM6000B</td>
<td>&nbsp;</td>
<tr>
<td>MicroPoint Manual Laser Illumination System</td>
<td>&nbsp;</td>
<td>Photonic Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>L06690</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E10521</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115836991</td>
<td>&nbsp;</td>
<tr>
<td>Plastic thread</td>
<td>&nbsp;</td>
<td>Any sewing supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Research Products International</td>
<td>A20090</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>Savant SPD131DDA</td>
<td>&nbsp;</td>
<tr>
<td>Vortexing mixer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Vortex Genie 2</td>
<td>&nbsp;</td>
<tr>
<td>Pressure injector</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

Ein zentrales Problem in der Entwicklungsbiologie ist es, den Ursprung der unzähligen Zelltypen, die in Wirbeltieren wie sie sich aus undifferenzierten Vorstufen entstehen abzuleiten. Die Forscher haben verschiedene Methoden der Linie Kennzeichnung, wie DiI Markierung 1 und Druck Injektion nachvollziehbar Enzyme 2 bis festzustellen Zellschicksal in späteren Stadien der Entwicklung in Modellsystemen eingesetzt. Die ersten Schicksal Karten im Zebrafisch (Danio rerio) wurden von iontophoretischen Injektion von Fluoreszenzfarbstoffen, wie Rhodamin Dextran montiert, in einzelne Zellen in diskreten Regionen des Embryos und Tracing der markierten Zelle das Schicksal im Laufe der Zeit 3-5. Während wirksam sind diese Methoden technisch anspruchsvoll und erfordern eine spezielle Ausrüstung nicht allgemein in Zebrafisch-Forschungsgruppen gefunden. In jüngster Zeit sind photoconvertable fluoreszierende Proteine, wie Eos und Kaede, die irreversibel von grünen Schalter auf rote Fluoreszenz bei UV-Licht ausgesetzt, sehen verstärkten Einsatz im Zebrafisch 6-8. Die optische Klarheit der Zebrafisch-Embryos und die relative Leichtigkeit der Transgenese haben diese besonders attraktiv Werkzeuge für die Linie Etikettierung gemacht und die Migration von Zellen in vivo 7 zu beobachten. Trotz ihrer Nützlichkeit, haben diese Proteine ​​auch einige Nachteile auf Farbstoff-vermittelte Linie Kennzeichnung Methoden verglichen. Der wichtigste ist die Schwierigkeit, die wir bei der Beschaffung von hoher 3-D-Auflösung während Photokonversion dieser Proteine ​​gefunden haben. In diesem Licht, vielleicht die beste Kombination aus Auflösung und Benutzerfreundlichkeit für Linie Kennzeichnung im Zebrafisch nutzt Käfig Fluorescein Dextran, einem Fluoreszenzfarbstoff, die zu einer Löschung Gruppe gebunden ist, dass Masken seine Fluoreszenz 9. Der Farbstoff kann dann &quot;uncaged&quot; (veröffentlicht von der Löschung Gruppe) innerhalb einer bestimmten Zelle mit UV-Licht von einem Laser-oder Quecksilber-Lampe, so dass Visualisierung der Fluoreszenz-oder Immundetektion. Im Gegensatz zu iontophoretischen Methoden können caged Fluorescein mit Standard-Einspritzvorrichtungen injiziert werden und uncaged mit einem Epifluoreszenzmikroskop mit einer Lochkamera 10 ausgestattet. Darüber hinaus erkennen Antikörper gegen Fluorescein nur die uncaged Form, und das Epitop überlebt Fixierung gut 11. Schließlich kann im Käfig Fluorescein mit einer sehr hohen 3-D-Auflösung aktiviert werden, besonders dann, wenn der Zwei-Photonen-Mikroskopie 12,13 eingesetzt. Dieses Protokoll beschreibt eine Methode der Linie Kennzeichnung von caged Fluorescein und Laser Uncaging. Subsequenctly ist uncaged Fluorescein gleichzeitig mit anderen Epitope wie GFP durch Markierung mit Antikörpern nachgewiesen.

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Dieses Protokoll zeichnet ein Weg, um Etiketten und Spurenelemente das Schicksal der kleinen Gruppen von Zellen Zebrafischembryonen mit UV-Uncaging der Käfig Fluorescein, durch whole mount Immunomarkierung, um das Signal aus dem uncaged Fluoreszein verstärken gefolgt.

Lineage Kennzeichnung von Zebrafisch-Zellen mit Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

Research

JoVE Journal

Biology

13.8K Aufrufe.  Vanderbilt University. Dieses Protokoll zeichnet ein Weg, um Etiketten und Spurenelemente das Schicksal der kleinen Gruppen von Zellen Zebrafischembryonen mit UV-Uncaging der Käfig Fluorescein, durch whole mount Immunomarkierung, um das Signal aus dem uncaged Fluoreszein verstärken gefolgt.

Serie: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Labeling Stammzellen mit fluoreszierenden Farbstoffen zur nicht-invasiven Detektion mit Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Forschung

Biologie

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Imaging Glykane in Zebrafisch-Embryonen durch metabolische Markierung und Bioorthogonale Click-Chemie

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Laser Ablation des Zebrafisch Vorniere auf Study Renal Epithelregeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

Bestimmung der Lipid Raft Partitionieren von Fluoreszenz-markierten Sonden in lebenden Zellen mittels Fluoreszenz-Korrelations-Spektroskopie (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phänotypische Analyse und Isolierung von murinen hämatopoetischen Stammzellen und Lineage-festgelegte Vorläuferzellen

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-Schädigung der Zebrafisch Embryonale Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluoreszenz-Imaging mit One-Nanometer-Genauigkeit (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Mikroinjektion in Zebrabärbling akuten Nierenschädigung zu Modell

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Traditional methods of assessing adipogenic differentiation are cheap and easy to use, but are not specific to changes in gene expression. We have developed an assay to quantify mesenchymal cell differentiation into mature adipocytes using a lineage specific marker. This assay has diverse applications across basic research and clinical medicine.

Visualisierung und Quantifizierung von mesenchymalen Zellen adipogenen Differenzierung Potenzial mit einer Linie spezifische Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying cellular and molecular mechanisms regulating lineage transitions of neural stem cells (NSCs) and eventual production of neurons and glia in the developing neuroepithelium remains unclear. Methods to trace NSC division patterns and map the lineage of clonally related cells have advanced dramatically. However, many contemporary lineage tracing techniques suffer from the lack of cellular resolution of progeny cell fate, which is essential for deciphering progenitor cell division patterns. Presented is a protocol using mosaic analysis with double markers (MADM) to perform in vivo clonal analysis. MADM concomitantly manipulates individual progenitor cells and visualizes precise division patterns and lineage progression at unprecedented single cell resolution. MADM-based interchromosomal recombination events during the G2-X phase of mitosis, together with temporally inducible CreERT2, provide exact information on the birth dates of clones and their division patterns. Thus, MADM lineage tracing provides unprecedented qualitative and quantitative optical readouts of the proliferation mode of stem cell progenitors at the single cell level. MADM also allows for examination of the mechanisms and functional requirements of candidate genes in NSC lineage progression. This method is unique in that comparative analysis of control and mutant subclones can be performed in the same tissue environment in vivo. Here, the protocol is described in detail, and experimental paradigms to employ MADM for clonal analysis and lineage tracing in the developing cerebral cortex are demonstrated. Importantly, this protocol can be adapted to perform MADM clonal analysis in any murine stem cell niche, as long as the CreERT2 driver is present.

Lineage Tracing and Clonal Analysis in Developing Cerebral Cortex Using Mosaic Analysis with Double Markers MADM

A protocol to perform lineage tracing and functional genetic analysis of candidate genes at a single cell level using mosaic analysis with double markers (MADM) is presented. MADM clonal analysis provides a quantitative framework to measure the proliferative behavior, cellular output, and lineage relationship of individual progenitors and their daughter cells.

Lineage Tracing und Clonal Analysis bei der Entwicklung von Zerebralparese mit Mosaikanalyse mit Doppelmarkern (MADM)

Beginning from a limited pool of progenitors, the mammalian cerebral cortex forms highly organized functional neural circuits. However, the underlying ...