Wir möchten S. Taneja-Bageshwar, K. Beifuss, S. Kedroske und HC danken. Hsiao für hilfreiche Diskussionen. Diese Arbeit wurde durch eine Anschubfinanzierung aus dem TAMHSC Department of Molecular and Cellular Medicine finanziert. Die Verbindung Umfang und drehende Scheibe wurden mit Mitteln der Abteilung und des TAMHSC Dekanat vorgesehen erworben. Einige Stämme wurden von den Caenorhabditis Genetics Center, das vom National Center for Research Resources finanziert wird. pRF4 (rol-6 (su1006)) war ein Geschenk von A. Fire.

1. Vorbereitung der DiI Fleck <ol><li> Bereiten Sie eine Stammlösung von 20 mg / mL DiI (Biotium, Inc., Hayward, CA) in DMF. DiI ist lichtempfindlich, so schützen DiI von Licht durch das Einwickeln in Folie. </li><li> Erstellen Sie ein Arbeitsverzeichnis Verdünnung von DiI durch Zugabe von 0,6 ul DiI Lager auf 399,4 ul M9 für jede Population. Dies sollte eine abschließende Arbeiten Verdünnung von 30 ug / mL DiI in M9. Dies kann bis zur Färbung mehrere Populationen gleichzeitig skaliert werden. Schild DiI von Licht durch Umwickeln des Rohres (s) in Folie. </li></ol> 2. Vorbereitung der Nematoden <ol><li> Verwenden Sie eine 60 mm Platte mit einer Bevölkerung von unberührten Nematoden. Wash Tiere von der Platte mit einer Lösung von 0,5% Triton X-100 in M9-Puffer durch vorsichtiges Schwenken Flüssigkeit in einer kreisförmigen Bewegung über die Oberfläche der Platte, alle Larven und ausgewachsene Tiere zu lockern. Transfer waschen in ein steriles 1,5 ml Tube. </li><li> Sofort-Spin-Down der Tiere bei 2000 rpm für 30 sek. Entfernen und so viel sup verwerfenernatant wie möglich, ohne die Masse der Tiere am Boden des Röhrchens. </li><li> Um die restlichen Triton X-100 zu reduzieren, spülen Tiere mit M9-Puffer, Spin und Überstand entfernen. Wiederholen Sie diesen Schritt einmal. </li><li> Add 400 ul arbeiten DiI-Lösung in M9 mit dem Rohr und kurz vortexen, um Tiere in der Lösung zu resuspendieren. </li><li> Schütteln Rohr bei 20 ° C horizontal für 3 Stunden bei 350 rpm in einem Licht-geschützten Umgebung. Wenn gewünscht, können die Tiere bis zu 16 Stunden für die Färbung inkubiert werden. </li><li> Zur Verringerung der Menge an ungebundenem Farbstoff, Spin-down der Tiere bei 2000 rpm für 20 sek. Entfernen und entsorgen Sie so viel Überstand wie möglich, ohne die Masse der Tiere. </li><li> Resuspendieren Tiere in 400 ul M9-Puffer und gießen Flüssigkeit auf eine Bakterien-freie Teil einer NGM-Agar-Platte mit OP50 E. ausgesät coli. Erlauben Tiere mindestens 30 Minuten zu erholen. Während der Erholungszeit der Tiere sollte weg zu kriechen aus der DiI Färbung flüssig und auf das Essen. Dieser Schrittreduziert Hintergrundfluoreszenz aus freien DiI. </li></ol> 3. Montage und Beachtung Proben <ol><li> Melt 4% Agar in Wasser unter Verwendung eines Autoklaven oder eine Mikrowelle. </li><li> Erstellen Sie wiederverwendbare Spacer, die verwendet werden, um gleichmäßige Dicke der Agar-Pad zu gewährleisten, kann durch Schichtung zwei Stücke von Labor-Band auf einem Glasträger. Machen Sie zwei spacer Dias insgesamt. </li><li> Vereinbaren Sie einen sauberen Objektträger aus Glas zwischen zwei spacer Dias. Pipette etwa 150 ul (vier Tropfen) von geschmolzenem 4% Agar auf die Mitte des sauberen Objektträger aus Glas. Schnell die geschmolzenen Agar mit einer zusätzlichen Folie in einem Agar-Pad zu bilden. Entfernen Sie vorsichtig den Deckel gleiten, halten Sie das Pad auf der Oberseite der Halterung schieben zentriert. </li><li> Pipette etwa 5 ul der Nematoden-Narkose (100 uM - 1 mM Levamisol, zum Beispiel) auf dem Pad. </li><li> Montieren von 8 bis 12 Tieren in der Narkose und mit einem Mikroskop Deckglas. </li><li> Beobachten Sie Tiere unter Verwendung einer Verbindung oder konfokalen Mikroskop mit mindestens einem 40x Objektivierung ausgestattet<html> ve und DsRed / TRITC (oder einem anderen kompatiblen)-Filter. Die Fluoreszenzanregung maximal DiI ist 549 nm und dessen Emissionsmaximum liegt 565 nm für gebundene Farbstoff (Biotium, Inc., Hayward, CA). </li></ol> 4. Repräsentative Ergebnisse DiI färbt die Nagelhaut von Wildtyp-und Mutanten-C elegans. Die Kutikula Oberfläche enthält Kreisringe durch umlaufende Rillen voneinander getrennt und in manchen Phasen, genannt Längsrippen alae. Jede Entwicklungsstufe hat Kutikula-Strukturen mit unterschiedlichen Zusammensetzungen 2. Ridges oder Furchen der beiden Nasenflügel und Kreisringe Fluoreszenz-Färbung, je nach Zusammensetzung der Oberfläche, während Larven und adulte Stadien und sichtbar bleiben bis zu einem Tag nach der Wiederherstellung mit dieser Methode. Hintergrund fluoreszierenden Flecken werden manchmal beobachtet (Abb. 2F, G), aber nicht routinemäßig (Abbildung 1, 2A-E, H, I). Alle Bilder wurden mit Spinning-Disk-konfokale oder, wenn vermerkt, Weitfeld (wf) Verbindung Mikroskopie genommen. ENT &quot;&gt; <img alt="Abbildung 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> Abbildung 1. DiI Flecken Nagelhaut und umweltfreundliche ausgesetzt Neuronen. L2 Bühne Tier. 630x Vergrößerung. Mosaic Bildteile wurden unter Verwendung iVision-Mac-Software (BioVision Technologies, Exton, PA). Die Bilder wurden trat mit Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). Balken = 10 um. DiI auch fluoreszierend Flecken amphid und phasmid sensorischen Neuronen im Kopf und Schwanz bzw. (Pfeile markieren einige). <img alt="Abbildung 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> Abbildung 2. DiI fluoreszierend Flecken C. elegans Schuppenschicht auf allen Stufen der post-embryonale Entwicklung. 630x Vergrößerung. Balken = 10 um. Die Färbung von Wildtyp-Tieren in A) L1; B) L2; C) Dauer; D) L3; E) L4 und F) adulte Stadien. Die Alen und ringförmige Grate sind fluoreszierend in L1 und dauer Tiere gefärbt (A, C). DiI Flecken ringförmige Grate in L2 Tiere(B). Ringförmigen Furchen Fleck in L3 und L4 Tiere (D, E). Die Furchen der Nasenflügel und Kreisringe sind bei erwachsenen Tieren (FH) gefärbt. Alae bestehen aus zwei, fünf, oder drei Rippen (in L1, Dauer, oder Erwachsener Tiere), dass die Länge des Tieres (Pfeile) 16 laufen zusammen. Kreisringe erstellen umlaufende Stege rund um das Tier (Pfeilspitzen, F und J). Die Kutikula der Erwachsenenbildung mutierten Tiere Display moderate Kutikula Organisation Mängel (GH). G) Ridges von Alen sind diskontinuierliche (wf). H) Überzählige alae Grate sind verschmolzen und verzweigt oder gegabelt (wf). Collagen-Gen-Mutanten weisen Alen und ringförmige Organisation Mängel (IJ). I) In transgenen Tieren überexprimierenden pRF4 (rol-6 (su1006)), Kämme alae liegen in einem Winkel zu der Länge des Tieres. J) Kreisringe in transgenen Tieren überexprimierenden pRF4 (rol-6 (su1006)) Display ein unregelmäßiges Muster. <img alt="Abbildung 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> Abbildung 3. External morphologischen Strukturen sind durch DiI Färbung beleuchtet. 630x Vergrößerung. Maßstabsbalken = 10 pm. DiI hebt auch andere äußere Merkmale, darunter A) erwachsene Hermaphrodit Vulva, B) erwachsenen männlichen Schwanz Strahlen und Lüfter, und C) Zwitter Tailspike (forked in dieser Mutante Hintergrund). <img alt="Abbildung 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> Abbildung 4. Cuticle länger dauert, bis mit DiI als umweltfreundliche ausgesetzt Neuronen Fleck. 630x Vergrößerung. Balken = 10 um. Nach zwei Stunden nach dem Färben sind amphid (nicht dargestellt) und phasmid sensorischen Neuronen (Pfeile) ausreichend gefärbt. Im Gegensatz dazu ist die Kutikula der jüngeren Tieren nur teilweise in Patches (Pfeilspitze) gefärbt. <table border="1"><tbody><tr><td> Waschen </td><td> Stain-Lösung (+ DII) </td><td> Inkubationszeit </td><td> Cuticle gebeizt </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 2 Stunden </td><td> nicht </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 3 Std. </td><td> nicht </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 2 Stunden </td><td> teilweise </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 3 Std. </td><td> ja </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 2 Stunden </td><td> teilweise </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 3 Std. </td><td> ja </td></tr></tbody></table> Tabelle 1. Kutikula Färbung unter verschiedenen Bedingungen. Various Inkubationslösungen und Zeiten wurden getestet, um Kutikula Färbung der Tiere zu optimieren. H 2 0, sterilem, destilliertem Wasser. Teilweise Färbung zeigt fleckige Färbung Larvencuticula (Abbildung 4), obwohl erwachsene Nagelhaut Flecken consistently.

<ol>
	<li>Cox, G. N., Kusch, M., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+of+Caenorhabditis+elegans:+its+isolation+and+partial+characterization.">Cuticle of Caenorhabditis elegans: its isolation and partial characterization.</a> The Journal of Cell Biology. 90, 7-17 (1981).</li><li>Cox, G. N., Staprans, S., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle+of+Caenorhabditis+elegans.+II.+Stage-specific+changes+in+ultrastructure+and+protein+composition+during+postembryonic+development.">The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development.</a> Dev. Biol. 86, 456-470 (1981).</li><li>Hall, D., Altun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans Atlas. , (2008).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> The C. elegans Research Community. , (2007).</li><li>Kramer, J. M., Johnson, J. J., Edgar, R. S., Basch, C., Roberts, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sqt-1+gene+of+C.+elegans+encodes+a+collagen+critical+for+organismal+morphogenesis.">The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis.</a> Cell. 55, 555-565 (1988).</li><li>Mende, N. v. o. n., Bird, D. M., Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dpy-13:+a+nematode+collagen+gene+that+affects+body+shape.">dpy-13: a nematode collagen gene that affects body shape.</a> Cell. 55, 567-576 (1988).</li><li>Blaxter, 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=Cuticle+surface+proteins+of+wild+type+and+mutant+Caenorhabditis+elegans.">Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 268, 6600-6609 (1993).</li><li>Costa, M., Draper, B. W., Priess, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+collagen+genes.+Expression+in+Caenorhabditis+elegans.">Cuticle collagen genes. Expression in Caenorhabditis elegans.</a> Trends Genet. 16, 21-27 (2000).</li><li>Kramer, J. M., French, R. P., Park, E. C., Johnson, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+rol-6+gene,+which+interacts+with+the+sqt-1+collagen+gene+to+determine+organismal+morphology,+encodes+a+collagen.">The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.</a> Mol. Cell Biol. 10, 2081-2089 (1990).</li><li>Thein, 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=Caenorhabditis+elegans+exoskeleton+collagen+COL-19:+an+adult-specific+marker+for+collagen+modification+and+assembly,+and+the+analysis+of+organismal+morphology.">Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.</a> Dev. Dyn. 226, 523-539 (2003).</li><li>McMahon, L., Muriel, J. M., Roberts, B., Quinn, M., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+dye-filling+of+sensory+neurons+in+Caenorhabditis+elegans.">Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.</a> J. Neurosci. Methods. 188, 58-61 (2010).</li><li>Singh, R. N., Sulston, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li></ol>

Wir haben nichts zu offenbaren.

Die DiI Färbung hier vorgestellte Methode ermöglicht eine relativ schnelle und bequeme Möglichkeit, um die Schuppenschicht in C. elegans zu visualisieren. Durch Wiederverwendung und Optimierung eines Verfahrens gemeinhin Bild umwelt ausgesetzt sensorischen Neuronen 15,17 verwendet wird, kann DiI verwendet werden, um Fluoreszenz-Färbung sowohl Alen und ringförmige Strukturen (Abbildungen 1 und 2) sowie der Vulva, männlichen Schwanz, und Hermaphroditen Tailspike werden (Abbildung 3). Wir haben fe...

<table border="1"><tbody><tr><td> Reagenzien </td><td> Synonyme </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> DiI </td><td> 1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine Perchlorat </td><td> Biotium, Inc. </td><td> 60010 </td><td> Lager Verdünnung: 20 mg / mL in DMF Gebrauchsverdünnung: 30 mg / mL </td></tr><tr><td> DMF </td><td> Dimethylformamid </td><td> Sigma-Aldrich, Inc. </td><td> D4551 </td><td></td></tr><tr><td> Triton X-100 </td><td> Octylphenoxypolyethoxyethanol </td><td> VWR International, LLC. </td><td> EM-9410 </td><td></td></tr><tr><td> M9 </td><td> 22mm KH 2 PO 4, 42mm Na 2 HPO 4, 86mm NaCl, 1 mM MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ Td&gt; <td> Nematode Wachstumsmedium </td><td> IPM Scientific, Inc. </td><td> 11006-501 </td><td> Kann folgenden NGM-Agar-Protokoll 18 hergestellt werden </td></tr><tr><td> Agar-Agar </td><td></td><td> EMD Chemicals, Inc. </td><td> 1,01614 </td><td> 4% in Wasser </td></tr><tr><td> Levamisol </td><td> Levamisol hydrochloride </td><td> Sigma-Aldrich, Inc. </td><td> 31742 </td><td> 100 pM - 1 mM Levamisol nach Bedarf </td></tr><tr><td> Objektträger </td><td></td><td> VWR International, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> Deckgläser </td><td></td><td> VWR International, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> Compound Rahmen </td><td></td><td> Carl Zeiss, Inc. </td><td> A1m </td><td> Verwenden Sie Ziele auf die Bedürfnisse des Experiments überein </td></tr><tr><td> TRITC oder anderen kompatiblen Filter </td><td> &amp;nbsp; </td><td> Chroma Technology Corp </td><td> 49005 </td><td> ET - DsRed (TRITC/Cy3) gesputterten Filter gesetzt </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

Die Kutikula von C. elegans ist eine sehr widerstandsfähige Struktur, die das Äußere des Tieres 1-4 umgibt. Die Nagelhaut schützt nicht nur das Tier aus der Umwelt, sondern bestimmt auch Körperform und spielt eine Rolle in Motilität 4-6. Mehrere Schichten von epidermalen Zellen abgesondert umfassen die Kutikula, darunter eine äußerste Lipid-Schicht 7. Umlaufende Stege in der Kutikula genannt Kreisringe Muster der Länge des Tieres und sind in allen Phasen der Entwicklung 8 vorhanden. Alae sind Längsrippen, die vorhanden sind während bestimmter Phasen der Entwicklung, darunter L1, Dauer, und adulte Stadien 2,9. Mutationen in Genen, die Kutikula Kollagen Organisation beeinflussen können alter Kutikula Struktur und tierischen Körper Morphologie 5,6,10,11. Während Kutikula Bildgebung mit Verbindung Mikroskopie mit DIC-Optik ist es möglich, aktuelle Methoden, die Highlight Kutikula Strukturen gehören FluoreszenzProzent Transgenexpression 12, Antikörper-Färbung 13 und Elektronenmikroskopie 1. Labeled Weizenkeimagglutinin (WGA) wurde ebenfalls verwendet, um Kutikula Glykoproteine ​​visualisieren, ist aber bei der Lösung von feineren Strukturen Kutikula 14 begrenzt. Die Färbung der Kutikula Oberfläche unter Verwendung von fluoreszierenden Farbstoff wurde beobachtet, aber nie im Detail 15 gekennzeichnet. Wir präsentieren eine Methode zur Nagelhaut in Live-C visualisieren elegans mit dem rot fluoreszierenden Farbstoff lipophil DiI (1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine Perchlorat), die üblicherweise in C verwendet elegans zu visualisieren umwelt ausgesetzt Neuronen. Diese optimierte Protokoll für DiI Färbung ist eine einfache, robuste Methode für hochauflösende Fluoreszenz-Visualisierung von Ringen, alae, Vulva, männlichen Schwanz, und Hermaphroditen Tailspike in C. elegans.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Wir präsentieren eine Methode zur Nagelhaut in Live-Visualisierung<em&gt; C. elegans</em&gt; Mit dem rot fluoreszierenden Farbstoff lipophil DiI (1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine Perchlorat), die häufig in Verwendung<em&gt; C. elegans</em&gt; Zu visualisieren umwelt ausgesetzt Neuronen. Mit diesem optimierten Protokoll werden Alen und ringförmige Strukturen Kutikula von DiI gefärbt und mit der Verbindung der Mikroskopie.

Visualisierung von Caenorhabditis elegans Cuticle Strukturen mit der lipophilen Vital Dye, DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

visualization-caenorhabditis-elegans-cuticular-structures-using

Research

JoVE Journal

Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K Aufrufe.  Texas A&M University System Health Science Center, College of Medicine. Wir präsentieren eine Methode zur Nagelhaut in Live-Visualisierung C. elegans Mit dem rot fluoreszierenden Farbstoff lipophil DiI (1,1 '-Dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine Perchlorat), die häufig in Verwendung C. elegans Zu visualisieren umwelt ausgesetzt Neuronen. Mit diesem optimierten Protokoll werden Alen und ringförmige Strukturen Kutikula von DiI gefärbt und mit der Verbindung der Mikroskopie.

Serie: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Messung Caenorhabditis elegans Life Span in 96 Well Mikrotiterplatten

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Forschung

Biologie

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Grundlegende<em&gt; Caenorhabditis elegans</em&gt; Methoden: Synchronisation und Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

der Anbau von<em&gt; Caenorhabditis elegans</em&gt; In drei Dimensionen im Labor

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI Perfusion als Methode zur vaskulären Visualisierung in<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Quantitative Ansätze zur Untersuchung zellulärer Strukturen und Organelle Morphologie in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Reinigung und Analyse von Caenorhabditis elegans Extrazelluläre Vesikel

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Verwendung von Caenorhabditis elegans zum Screening auf gewebespezifische Chaperon-Interaktionen

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automatisierung der Aggregatquantifizierung bei Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Kleine Extraktion von Caenorhabditis elegans Genomischer DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...

Visualisierung von Caenorhabditis elegans Cuticle Strukturen mit der lipophilen Vital Dye, DiI

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Messung Caenorhabditis elegans Life Span in 96 Well Mikrotiterplatten

Grundlegende

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

der Anbau von

DiI Perfusion als Methode zur vaskulären Visualisierung in

Quantitative Ansätze zur Untersuchung zellulärer Strukturen und Organelle Morphologie in Caenorhabditis elegans

Reinigung und Analyse von Caenorhabditis elegans Extrazelluläre Vesikel

Verwendung von Caenorhabditis elegans zum Screening auf gewebespezifische Chaperon-Interaktionen

Automatisierung der Aggregatquantifizierung bei Caenorhabditis elegans

Kleine Extraktion von Caenorhabditis elegans Genomischer DNA