Wir erkennen die Caenorhabditis Genetics Center for C. elegans-Stämme. Finanzielle Unterstützung durch die Terry Fox Research Institute bereitgestellt. Wir unterstützen EP aus der Rolande und Marcel Gosselin Graduiertenstipendium und der CIHR / FRSQ Ausbildungsförderung in der Krebsforschung FRN53888 der McGill Integrated Cancer Research Training Program gewährt erkennen auch.

1. Vorbereitung der Reagenzien <ol><li> Herstellung von Medien für C. elegans Wachstum (in diesem Fall, Tiere Wildtyp und FLCN-1 (ok975) mutierten Tiere). <ol><li> Bereiten Geändert Youngren nur Bactopepton (MYOB) Trockenmischung, die 5,5 g Tris HCl, 2,4 g Tris-Base, 31 g Bactopetone, 20 g NaCl und 0,08 g Cholesterin. Gut mischen mit Schütteln. Anmerkung: Diese Mischung ist ausreichend, um 10 l MYOB Medium herzustellen. HINWEIS: normalem Wachstumsmedium (NGM) Platten könnte statt MYOB Platten 27 verwendet werden. Jedoch sollte die gleiche Art von Platten für gültige Vergleiche zwischen Stämmen und zwischen unabhängigen wiederholt verwendet werden. In diesem Protokoll haben wir nur MYOB Platten. </li><li> Bereiten Sie 1 l MYOB Medium durch Zugabe von 6 g MYOB Trockenmischung, 17 g Agar und auf 1 L mit H 2 O und Autoklav für 45 Minuten bei 122 ° C. Platten gießen und lassen bei Raumtemperatur trocken für 2 Tage. </li><li> Unter Einhaltung steriler Techniken impfen 100 ml cultuWieder LB-Brühe-Medium mit E. coli OP50 Bakterien und wachsen O / N bei 37 ° C. </li><li> Seed MYOB Platten mit E. coli OP50 Bakterien. Lassen Sie es bei Raumtemperatur für 48 Stunden wachsen. </li></ol></li><li> Vorbereitung für die Gen-Downregulation mit RNAi <ol><li> Bereiten MYOB RNAi-Zuführung Platten. Gehen Sie wie in den Schritten 1.1.1 und 1.1.2. Nach dem Autoklavieren Das Medium auf etwa 55 ° C abkühlen gelassen und mit 1 ml 1 M Isopropyl β-D-1-thiogalactopyranosid (IPTG) und 1 ml 100 mM Ampicillin. </li><li> Streak E. HT115 coli Bakterien, die mit einem Plasmid, das entweder Steuerung EV oder FLCN-1 spezifische RNAi auf Ampicillin (50 ug / ml) / Tetracyclin (15 ug / ml) Agarplatten drückt umgewandelt. Kultur wachsen O / N bei 37 ° C. </li><li> Wählen Kolonien und impfen E. coli HT115 Bakterien mit einem Plasmid, das entweder Steuer EV oder FLCN-1 spezifische RNAi und wachsen Kultur in LB / Ampicillin (50 ug / ml) mittel- und O / N bei 37 &amp; # drückt transformiert176; C. </li><li> Samen Platten mit E. coli HT115 EV Bakterien oder HT115 FLCN-1 RNAi Bakterien. Hinweis: Bewahren Sie Platten 48 Stunden bei Raumtemperatur vor der Verwendung. Wenn RNAi durch Einspeisen in Wirk, andere Verfahren der Abgabe der ds-RNA 28. </li></ol></li><li> Induziert oxidativen Stress mit Paraquat (PQ): <ol><li> Vorbereitung M9 Puffer durch Mischen von 3 g KH 2 PO 4, 6 g Na 2 HPO 4, 5 g NaCl und 0,25 g MgSO 4 · 7 H 2 O. Bringen Sie bis zu 1 L mit destilliertem Wasser. Autoklaven-Lösung für 45 min bei 122 ° C und zu speichern Flaschen bei RT. </li><li> Am Tag des Assays, bereiten Sie die M9 / PQ-haltigen Lösung. Für dieses Protokoll verwenden 100 mM PQ (0,1028 g PQ in 4 ml M9-Puffer füllt ein ganzes 96-Well-Platten mit 40 ul M9 / PQ pro Vertiefung). ACHTUNG: PQ ist eine gefährliche und hochgiftige Verbindung. Griff nach entsprechenden Richtlinien. HINWEIS: Beim ersten Ausführen eine neue sZug oder ein neuer Zustand, ist es empfehlenswert, um das Überleben in steigenden Konzentrationen von PQ um das beste Konzentration, um die Tiere in 7-10 h zu töten verwenden bestimmen, zu untersuchen. </li></ol></li></ol> 2. Herstellung von Age-synchronisiert L4 Bevölkerung <ol><li> Transfer 20 gravid Erwachsenen Hermaphroditen auf eine frische Platte mit MYOB E. ausgesät coli OP50 Bakterien. Vorbereitung mehrere Platten auf der Basis der Anzahl der Würmer bei den Assay benötigt. </li><li> Lassen Sie sie Eier für 6 Stunden und dann mit einem Platindraht Wurm Pick entfernen Sie die Mütter legen. Überprüfen Sie die Platten unter dem Mikroskop, um die Anzahl der gelegten Eier zu bewerten. </li><li> Lassen Sie die Eier schlüpfen und wachsen bei 20 ° C für 48 Stunden. Zu dieser Zeit sind die Tiere in einem späten L4 / junger Erwachsener Bühne. Wählen gleichen Stufe Tiere und Transfer zum PQ Lösung haltigen Brunnen. Hinweis: Da 48-stündiger Inkubation nur auf Mutanten, die ähnlich zu den Wildtyp-Tieren wachsen, ist es wichtig, zu überwachen EntwicklungsRate der neu getesteten Mutanten, um sicherzustellen, es erfüllt die Entwicklungsrate Wildtyp, sonst sollten andere Zeitleisten verwendet werden. </li><li> Alternativ können Sie Synchronisationstechniken. Generieren synchronisiert Bevölkerung von L1-Larven Würmer mit Hypochlorit-Lösung (25 ml Wasser, 125 ml von 5,25% Natriumhypochlorid, 50 ml von 4 M NaOH; wickeln Flasche mit Aluminiumfolie vor Licht zu isolieren). Für diesen Test ist es nicht empfehlenswert, da intensive Bleich könnte das Verhalten der Tiere während des Assays beeinflussen. VORSICHT: Gehen Sie Hypochlorit-Lösung nach den Richtlinien. HINWEIS: Bei der Verwendung von RNAi, um nach unten zu regulieren das Gen von Interesse, die Hermaphroditen zu synchronisieren, wie in Kapitel 2 beschrieben, mit der Ausnahme von der Übertragung von Müttern auf RNAi-Platten mit den spezifischen RNAi Bakterien geimpft. Wenn der Zuschlags mit der RNAi schwach ist, wird Fütterung für mehrere Generationen empfohlen. </li></ol> 3. Durchführung der oxidativen Stressresistenz Assay &lt;/ P&gt; <ol><li> Pipette 40 ul der 100 mM PQ-Lösung (frisch zubereitet) auf jede Vertiefung einer 96-Well-Platte. Verwenden Sie mindestens 12 Wells als repliziert jeder Bedingung (Behandlung, mutant, etc.). Unter Verwendung einer Platindraht-Wurm Pick, übertragen 5-8 L4-Larven Tiere jeden gut, die die Startzeit und die Endzeit. </li><li> Beim Start eine weitere Bedingung, beachten Sie die Startzeit und die Endzeit. Legen Sie die Platte bei 20 ° C in der Inkubationszeit zwischen toten Tieren eine Stunde später die Übertragung der Würmer und Scoring von der Startzeit. </li><li> Verwenden des Binokular, zählen Überlebens jeden hr. Die Platte Schütteln Sie vor dem Start zu zählen. Geben Sie Würmer, die sich nicht bewegen, auch nach dem Absetzen einer Licht von hoher Intensität, wie tote Tiere. Auch die Anzahl der Tiere am Leben. HINWEIS: Mit Blick auf Schneckenform, Schwänze und Kopfbewegung bei hoher Vergrößerung, um festzustellen, tote Würmer lebendig. </li><li> Wiederholen Sie diesen Schritt jede Stunde, bis die meisten Würmer sind tot. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Bestimmung der Überlebensprozent zu jedem Zeitpunkt <ol><li> Berechnen Sie die Gesamtzahl der Tiere pro Bedingung, indem Sie die Gesamtzahl der Tiere, um jedes über gut. Ignorieren Sie die Tiere, die beschädigt sind oder bei der Übertragung getötet. </li><li> Für jeden Zeitpunkt berechnen die Gesamtzahl der toten Tieren pro Bedingung (Summe der toten Tiere in den 12 Vertiefungen). Für jeden Zeitpunkt berechnen Prozentsatz des Todes (Anzahl der toten Tiere dividiert durch die Gesamtzahl der Tiere und multipliziert mit 100) und den Prozentsatz des Überlebens (100 minus Prozent Tod). </li><li> Wiederholen Sie diesen Test mindestens 3 unabhängigen Zeiten. </li></ol>

<ol>
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The authors declare that they have no competing financial interests.

C. elegans ist ein attraktives Modellorganismus genetisch oxidative Stressresistenz in vivo zu untersuchen, da es leicht gezüchtet werden können, und führt zu einer Vielzahl genetisch identischer Nachkommenschaft schnell. Mehrere Methoden zur oxidativen Stress Widerstand zu messen, wurden zuvor beschrieben und sie sind auf die Ergänzung von Kulturplatten mit verschiedenen ROS Quellen wie PQ, Rotenon, H 2 O 2 und Juglon 25,26,29-32 basiert. Hier beschreiben wir ein P...

A reference was corrected in the publication of <a href="http://www.jove.com/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

Erratum: Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

In Eukaryoten ist die oxidative Phosphorylierung, die sich in der Elektronentransportkette der Mitochondrien der Haupttreiber der Energieproduktion in Form von ATP. Reaktive Sauerstoffspezies (ROS) sind ein natürliches Nebenprodukt des Prozesses. Trotz ihrer wichtigen Rolle als Signalmoleküle, können übermäßige ROS auf DNA-Schäden, Protein-Carbonylierung und Lipidoxidation führen. Ein Ungleichgewicht zwischen ROS-Produktion und Entgiftung verursacht oxidativen Stress, was zu Energiemangel, Zellschäden führt, und löst den Zelltod 1,2. Oxidativer Stress trägt zur Alterung und auf die Entwicklung von vielen lebensbedrohlichen Krankheiten wie Krebs, Diabetes, Herz-Kreislauf- und neurodegenerative Erkrankungen 3-9. Zellen enzymatischen und nicht-enzymatischen Abwehrstrategien entwickelt, um die ordnungsgemäße ROS Niveau zu halten und ihre Bestandteile vor oxidativen Schäden schützen 1,2. Superoxid-Dismutase (SOD) Enzyme wirken Erste convert Superoxid zu H 2 O 2, die später in Wasser umgewandelt wird von Katalase oder Peroxidase-Enzyme. Nicht enzymatischen Abwehrstrategien beinhalten meist Moleküle, die schneller mit ROS im Vergleich zu zellulären Makromolekülen, zum Schutz wesentlicher Zellkomponenten reagieren. Trotz der schützende Rolle von ROS Entgiftungsenzyme, einige ROS-Moleküle entweichen die antioxidativen Abwehrmechanismen und führen zu oxidativen Schäden. Erkennung, Reparatur und Abbau der beschädigten zellulären Komponenten sind wesentliche Verteidigungsstrategien während oxidativem Stress 1,2. Signalwege in Stressresistenz und speziell oxidativem Stress beteiligt sind hoch evolutionär konservierte 10,11. Im Gegensatz zu Zellkulturexperimenten, wo organismal Bedingungen nur teilweise wiedergegeben werden, die Untersuchung von oxidativem Stress in Modellorganismen 12,13 hat eine große Bedeutung. C. elegans ist ein freilebenden Nematoden, die einfach und kostengünstig sein kann culnissen auf einem Bakterienrasen auf Agar-Medien. Es ist klein (ca. 1 mm in der Länge) und in der Regel wächst als selbstDünge Zwitter, die genetische Manipulationen erleichtert. Es hat eine schnelle Lebenszyklus und eine hohe Fortpflanzungsfähigkeit, die rund 300 Nachkommen pro Generation, so dass es ein leistungsfähiges Werkzeug, um groß angelegte genetische Screens 14 durchzuführen. C. elegans Genom vollständig sequenziert und 40-50% der Gene vorhergesagt werden, um Homologe des humanen krankheitsassoziierten Genen 15-18 sein. Der Knockdown von Genen von Interesse unter Verwendung von RNAi ist eine schnelle und einfache in C. elegans. Gene Herunterregulierung könnte durch Füttern von Tieren der E. erreicht werden coli-Bakterien, die ein Plasmid, das das doppelsträngige RNA, die die mRNA von Interesse 19 zielt exprimieren beherbergen. Daher hat die Bestimmung der Genfunktion mit Groß RNAi-Screens großen Einfluss auf das Verständnis der menschlichen Krankheiten, darunter Krebs 20,21. Studium der oxidative Stressresistenz in C. elegans haben zur Identifizierung von konservierten Mechanismen der Resistenz gegen oxidative Belastung 13,22 geführt. Einige Wege identifiziert normale Wege, die Langlebigkeit und Beständigkeit zu modulieren anderen Belastungen als auch, wie Hypoxie, Wärme und osmotischen Stress. Diese Wege sind die Insulin-Signal, TOR-Signalisierung und Autophagie. Weitere wichtige Signalwege beinhalten Entgiftung von ROS wie Superoxid-Dismutase Enzyme und Enzyme Katalase, oder in Schaden zu reparieren, wie Hitzeschock und Chaperon-Proteine ​​11,13,22. Dieses Protokoll beschreibt, wie die Widerstandsfähigkeit gegenüber oxidativem Stress von C. bestimmen elegans in Flüssigkeit. Wir verwendeten FLCN-1 (ok975) und Wildtyp-Tieren, das Protokoll zu zeigen, da wir zuvor in C gezeigt, eine erhöhte Resistenz gegenüber oxidativem Stress bei Verlust der FLCN-1 (ok975) elegans 23. Wir haben auch gezeigt, dass dieser Widerstand hängtauf AMPK und Autophagie, einen Signalisierungs Achse, die zelluläre Bioenergetik verbessert und fördert die Stressresistenz 23. PQ ist eine oxidative Stressfaktor, der mit der Elektronentransportkette an reaktiven Sauerstoffspezies produzieren 24 stört. Das gleiche Assay könnte angepasst werden und andere ROS Quellen oder ROS erzeugende Verbindungen könnten als H 2 O 2 und Rotenon verwendet werden, so. Ähnliche Tests wurden auf Platten, wo niedrige Konzentrationen von PQ verwendet 25,26 entwickelt. Der Vorteil dieses Tests ist, dass es sehr schnell ist, und die Ergebnisse können in einem Tag erreicht werden. Zusätzlich ist das Gesamtvolumen der Flüssigkeit verwendet, um den oxidativen Stress Resistenzassay in Platten mit 96 Vertiefungen durchgeführt gering, verglichen mit der zur Bestimmung PQ-enthaltenden Platten herzustellen Volumens. Daher ist die Menge des verwendeten PQ im flüssigen Assay gering ist, was den Assay macht preiswerte und begrenzt die Erzeugung von Giftmüll. , Grenzen dieses Tests im Vergleich zu Plattentests sind jedoch die lack von Nahrungsmitteln im flüssigen Assay und der geringeren Konzentration an Sauerstoff in flüssiger, im Vergleich zu Luft. Dies sind wichtige Faktoren, die in einigen Fällen die Ergebnisse beeinflussen. Daher bestätigt die Reproduzierbarkeit mit anderen Methoden von oxidativem Stress Widerstand wird empfohlen, die Ergebnisse in diesem Test erhalten unterstützen.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agar bacteriological grade</td>
			<td>Multicell</td>
			<td>800-010-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological peptone</td>
			<td>Oxoid</td>
			<td>LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride biotechnology grade</td>
			<td>Bioshop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma</td>
			<td>C8503-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure tris hydrochloride</td>
			<td>Invitrogen</td>
			<td>15506-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris aminomethane</td>
			<td>Bio Basic Canada Inc</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202185A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Bio Basic Inc.</td>
			<td>8013-01-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Aldrich chemistry</td>
			<td>856177-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60x15mm</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10ml</td>
			<td>Fisher</td>
			<td>1367520</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>G-Biosciences</td>
			<td>RC-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M-5921</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Bioshop</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Discovery v8 stereo zeiss microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear microtiter plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flcn-1 RNAi source</td>
			<td colspan="2">Ahringer Library</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates

Oxidativer Stress, der die Folge eines Ungleichgewichts zwischen der Erzeugung und Entgiftung von reaktiven Sauerstoffspezies ist, ist ein wesentlicher Faktor, um chronische Humanerkrankungen, kardiovaskulären und neurodegenerativen Erkrankungen, Diabetes, Altern und Krebs. Daher ist es wichtig, oxidativen Stress nicht nur in Zellsystemen als auch mit ganzen Organismen zu studieren. C. elegans ist eine attraktive Modellorganismus, um die Genetik von oxidativem Stress Signaltransduktionswege, die sehr evolutionär konserviert sind zu studieren. Hier stellen wir ein Protokoll, um oxidativen Stress Widerstand in C messen elegans in Flüssigkeit. Kurz gesagt, ROS-induzierenden Reagenzien wie Paraquat (PQ) und H 2 O 2 in M9-Puffer und Lösungen werden in die Vertiefungen einer Mikrotiterplatte mit 96 Vertiefungen aliquotiert. Synchronisiert L4 / jungen Erwachsenen C. elegans Tiere werden zu den Vertiefungen transferiert (5-8 Tiere / Vertiefung) und das Überleben wird gemessenjede Stunde, bis die meisten Würmer sind tot. Bei der Durchführung eines oxidativen Stressresistenz-Test unter Verwendung einer niedrigen Konzentration von Stressfaktoren in Platten, Alterung könnte das Verhalten der Tiere auf oxidativen Stress, was zu einer falschen Interpretation der Daten führen könnten, zu beeinflussen. In dem hier beschriebenen Assay Dieses Problem ist jedoch nicht wahrscheinlich, da nur L4 / junge Tiere verwendet werden. Darüber hinaus ist dieses Protokoll kostengünstig und Ergebnisse werden in einem Tag, die diese Technik attraktive für genetische Screens rendert erhalten. Insgesamt wird dies dazu beitragen, um oxidativen Stress Signalübertragungswege, die in bessere Charakterisierung des oxidativen Stress-assoziierten menschlichen Erkrankungen übersetzt werden konnte verstehen.

Vergleicht man Wildtyp C. elegans zu FLCN-1 (ok975) mutierten Tiere Hier haben wir 100 mM PQ, um den Widerstand von Wildtyp-C zu bestimmen elegans Tieren im Vergleich zu FLCN-1 (ok975) von dem gezeigt wurde auf oxidativen Stress, Hitze und Anoxie 23 zu widerstehen. Nach 4 Stunden der Behandlung, 48,3% des Wildtyp-überlebt, im Vergleich zu 77,8% Überlebensrate in FLCN-1 (ok975) Tiere. Wie erwartet, FLCN-1 (ok975) muti...

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Mess Oxidativer Stress Widerstandsrat<em&gt; Caenorhabditis elegans</em&gt; In 96-Well-Mikrotiterplatten

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

measuring-oxidative-stress-resistance-caenorhabditis-elegans-96-well

Research

JoVE Journal

Biology

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

15.3K Aufrufe.  McGill University. C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates. 

Serie: Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Messung Caenorhabditis elegans Life Span auf festen Medien

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Forschung

Biologie

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Isolation und In-vitro- Aktivierung von Caenorhabditis elegans Sperma

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Zeitraffer-Mikroskopie der frühen Embryogenese in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice-Cap: Ein Verfahren zur Züchtung von Arabidopsis Und Tomatenpflanzen in 96-well-Platten für High-Throughput-Genotypisierung

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Prostaglandin Extraktion und Analyse in<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Analyse von oxidativem Stress in Zebrafischembryonen

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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 ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Messung von Sauerstoff Verbrauchsmengen in intakten Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...