Diese Arbeit wurde von einem Piloten Auszeichnung des Nathan Shock Center (P30 AG13283) und R01 zu gewähren (R01 AG028516) aus dem National Institute on Aging (NIA) auf A.-LH unterstützt

<html> 1. Herstellen einer Eichkurve für die Bestimmung von Bakterien-Konzentrationen Dieser Abschnitt beschreibt eine Methode zur Abschätzung der Bakterien-Konzentration für einen bestimmten Bakterienstamm (zB OP50, HT115), die als Nahrungsquelle für Diätetische Einschränkung Experimente in C verwendet werden elegans. Separate Kalibrierkurven sollten für jeden Bakterienstamm verwendet eingerichtet werden. <ol><li> Impfen einer einzelnen Kolonie von E. coli OP50 Bakterien aufgenommen von einem frischen Streifen von Bakterien auf LB-Agar in 3 ml flüssiges LB-Brühe. </li><li> Legen Sie OP50 Kultur in 37 ° C Schüttler und lassen Bakterien wachsen über Nacht. </li><li> Bereiten Sie serielle 2-fache Verdünnungen der Bakteriensuspension aus der Übernacht-Kultur (2x, 4x, 8x, 16x, 32x). </li><li> Die Extinktion (optische Dichte) von jeder Verdünnung bei 600 nm mittels Spektralphotometer in dreifacher Ausfertigung. </li><li> Weitere verdünnen jeder Bakteriensuspension 1.000.000-fache (1000-facher Verdünnung zweimal). </li><li> Verbreiten Sie 0,4 ml Bakteriensuspension jeder Verdünnung gleichmäßig auf einem 100 mm LB-Agar-Platte. </li><li> Legen LB-Platten mit OP50 in einem 37 ° C Inkubator für 18-24 Stunden. </li><li> Zählen Sie die Anzahl der Bakterienkolonien auf jeder Platte gewachsen. Die Kolonie zählt werden verwendet, um KBE (Kolonie bildende Einheit) pro ml für jede Probe berechnet werden. </li><li> Die Beziehung zwischen der gemessenen OD 600 Werte und OP50 Bakterien-Konzentrationen können durch Auftragen von OD 600 als Funktion der Konzentration der Bakterien (KBE / ml) und eine lineare Regressionsanalyse (Abbildung 1) festgestellt werden. </li></ol> Solutions: <table border="1"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td > 10 g </td><td> Bacto-Trypton </td></tr><tr><td> 5 g </td><td> Hefeextrakt </td></tr><tr><td> 10 g </td><td> NaCl </td></tr></tbody></table> Add entionisiertem Wasser auf 1L. Autoklaven und bei Raumtemperatur lagern. 2. Herstellung von festen Platte für die Ernährung Einschränkung (SDR)-Platten Dieser Abschnitt beschreibt die Herstellung von Platten für den Einsatz in der Fest-Platte für die Ernährung Einschränkung (SDR) Experimente. Normalerweise betrachten wir eine Bakterien-Konzentration von 1 x 10 11 KBE / mL als ad libitum-Fütterung Zustand und 1 x 10 8 KBE / ml als optimal DR Fütterung Voraussetzung für diese Methode. Allerdings können 4-6 verschiedene Bakterien-Konzentrationen benötigt, um eine vollständige Dosis-abhängige Beziehung zum SDR und ihre Antworten zu etablieren. <ol><li> Impfen einer einzelnen Kolonie von E. Coli Bakterien OP50 in 3 ml flüssiges LB-Brühe. </li><li> Setzen Sie den OP50 Kultur in 37 ° C Schüttler und ermöglichen die Bakterien für 6-8 Stunden wachsen. </li><li> Übertragen Sie die OP50 Kultur zu einer neuen Kolben mit 500 mL LB-Bouillon, und platzieren Sie den OP50 Kultur wieder in 37 ° C Schüttler über Nacht. </li><li> Messen Sie die OD 600 der OP50 Kultur, um die Bakterien-Konzentration (die Konzentration wird anhand der Kalibrierkurve in Teil 1 erzeugt) zu bestimmen. Geeignete Verdünnungen können für eine genaue Messung der Absorption erforderlich sein. </li><li> Pellet OP50 Bakterien durch Zentrifugation bei 1.500 g für 20 Minuten. </li><li> Resuspendieren Bakterien zu einer Konzentration von 1 x 10 12 KBE / mL. </li><li> Bereiten Sie 10 mL Bakterienkultur in sechs verschiedenen Konzentrationen: 1 x 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 cfu / mL. </li><li> Pipette 0,2 ml Bakterienkultur in der Mitte eines 60 mm verfestigt NGM Platte. Berühren Sie die Oberfläche der Platte mit Pipettenspitze, wie Kerben auf der Oberfläche erlauben Würmer bohren sich in die Agarplatten. </li><li> Legen Platten in 37 ° C Inkubator für 1 Stunde. </li><li> Übernehmen 10 &amp;mgr; l von 100mg/ml Carbenicillin und 10 ul 50mg/ml Kanamycin zu jeder Platte das Wachstum der Bakterien zu verhaften. </li><li> Shop-Platten mit unterschiedlichen Konzentrationen von Antibiotika-abgetöteten Bakterien bei 4 ° C für den zukünftigen Gebrauch (bis zu 1 Monat). Es ist wichtig für die Vorbereitung und speichern genug Platten für die gesamte geplante Experiment mindestens 1-2 Tage im Voraus. Mit der gleichen Charge von Platten für die gesamte Experimenten vorzuziehen ist. </li><li> Um zu überprüfen, die Lebensfähigkeit der Bakterien, kratzen einige Bakterien aus der SDR-Platten und breitete sie auf einem leeren NGM-Platte mit Carbenicillin und Kanamycin. Die Platte wird in 37 ° C Inkubator für 6-8 Stunden und dann für das Wachstum der Bakterien (kein Wachstum von Bakterien zu beachten) zu überprüfen. </li></ol> Lösungen <table border="1"><tbody><tr><td colspan="2"> Nematode Growth Media (NGM), 1L: </td></tr><tr><td > 3 g </td><td> NaCl </td></tr><tr><td> 2,5 g </td><td> Bacto-Pepton </td></tr><tr><td> 20g </td><td> Agar </td></tr></tbody></table>ove_content &quot;&gt; Autoklaven für 40 Minuten abkühlen lassen und auf 65 ° C, dann hinzuzufügen: <table border="1"><tbody><tr><td > 1 mL </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 mL </td><td> 1 M CaCl 2 </td></tr><tr><td> 1 mL </td><td> 5 mg / ml Cholesterin </td></tr><tr><td> 25 mL </td><td> 1 M Kaliumphosphat, pH 6,0 </td></tr></tbody></table> Unmittelbar pour Flüssigkeit NGM auf 60 mm x 15 mm Petrischalen in 10 ml Aliquots 3. Einrichten SDR Experimente Für die meisten der SDR-Fächer, wie z. B. Lebensdauer Analyse und Fruchtbarkeit Profiling, Erstellung eines Alters-synchronisierte Bevölkerung von Würmern ist unerlässlich. Für andere Studien, einfach übertragen Würmer aus einer regelmäßigen OP50 Platte ein SDR Platte auf die diätetische Einschränkung zu initiieren. In diesem Abschnitt ist ein Protokoll zur Einrichtung einer Lebensdauer-Analyse für SDR Tiere als Beispiel beschrieben. <ol><li> Verwenden Sie ein Platin-Wurm-Picker auf 20-30 reproduktiv aktiven Erwachsenen auf mehrere regelmäßige NGM-Platten mit OP50 zu übertragen. Abhängig von der Anzahl der Eier für die Experimente benötigt wird, kann mehr Erwachsene benötigt werden. </li><li> Verlassen Sie die Platten bei 20 ° C für 4 Stunden, damit der Eiablage. </li><li> Entfernen Sie die Erwachsenen von der Platte. Die Platten sollten visuell auf Eier überprüft werden. </li><li> Verlassen Sie die Platten bei 20 ° C und ermöglichen die Nachkommen in die Late-L4/day 1 Erwachsener Bühne zu entwickeln. Normalerweise dauert dies 3 Tage N2 wilden Arten Würmer bei 20 ° C. </li><li> Übertragen einer geeigneten Anzahl von Tag 1 Erwachsenen auf SDR Platte auf die diätetische Einschränkung zu initiieren. </li><li> Für eine Lebensdauer-Analyse, die Einrichtung eines insgesamt von 6-8 Platten mit 10-20 Würmer pro Platte für jeden SDR Zustand. </li><li> Ergebnis der Lebensfähigkeit eines jeden Wurms alle 2 Tage, bis alle Würmer gestorben. Es ist wichtig, dass Würmer an die frische SDR-Platten übertragen werden jedes 12-48 Stunden in Anspruch Entzug von Nahrung zu vermeiden. </li></ol> 4. Repräsentative Ergebnisse: In Abbildung 1 ist ein Diagramm einer repräsentativen Eichkurve für die Schätzung der OP50-Konzentration in Bezug auf OD 600. Die Gleichung von Liner Regressionsanalyse erzeugt wird, kann für alle zukünftigen Berechnungen der OP50 Bakterien Konzentrationen eingesetzt werden. In Abbildung 2 ist ein Beispiel zur Untersuchung der Wirkung von chronischen SDR-Behandlungen auf die mittlere Lebensdauer der verschiedenen Stämme Wurm. Die blaue Linie stellt eine normale Dosis-abhängige Reaktion von Wildtyp-Würmer SDR. Die rote Linie zeigt an, dass die Lebensdauer Wirkung von SDR teilweise von DRR-2 oe unterdrückt, während daf-16 (mu86) hat keine Auswirkung auf SDR-induzierte Laufzeitverlängerung. Detail-Methode für die Durchführung von Lebensdauer-Analyse in C. elegans ist in der bisherigen Literatur 1-3 beschrieben. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Repräsentatives Ergebnis einer Eichkurve für die Abschätzung der E. Coli OP50 Konzentration. OP50-Konzentration als Funktion der gemessenen OD 600-Werte aufgetragen. Fehlerbalken = SD Übernachtung Bakterienkultur wurde 1.5x, 2x, 4x, 8x, 16x, 32x, bevor Extinktionsmessung verdünnt und weiter verdünnt 1.000 x zweimal, bevor cfu Messung. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Repräsentatives Ergebnis einer C. elegans Lebensdauer Experiment verglichen Wildtyp N2 Tiere (blau) mit zwei verschiedenen Mutanten (grün und rot) in Reaktion auf SDR (Abbildung von Ching et al. 4). Fehlerbalken = SEM

<ol>
	<li>Hsu, A. L., Murphy, C. T., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+aging+and+age-related+disease+by+DAF-16+and+heat-shock+factor.">Regulation of aging and age-related disease by DAF-16 and heat-shock factor.</a> Science. 300, 1142-1145 (2003).</li><li>Kenyon, C., Chang, J., Gensch, E., Rudner, A., Tabtiang, R. <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+mutant+that+lives+twice+as+long+as+wild+type.">A C. elegans mutant that lives twice as long as wild type.</a> Nature. 366, 461-464 (1993).</li><li>Apfeld, J., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lifespan+by+sensory+perception+in+Caenorhabditis+elegans.">Regulation of lifespan by sensory perception in Caenorhabditis elegans.</a> Nature. 402, 804-809 (1999).</li><li>Ching, T. T., Paal, A. B., Mehta, A., Zhong, L., Hsu, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=drr-2+encodes+an+eIF4H+that+acts+downstream+of+TOR+in+diet-restriction-induced+longevity+of+C.+elegans.">drr-2 encodes an eIF4H that acts downstream of TOR in diet-restriction-induced longevity of C. elegans.</a> Aging Cell. 9, 545-557 (2010).</li><li>Weindruch, R., Walford, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Retardation of Aging and Disease by Dietary Restriction. , (1988).</li><li>Masoro, E. 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Diätetische Einschränkung (DR) ist bekannt, dass Lebensdauer zu erhöhen und das Einsetzen der verschiedenen altersbedingten Erkrankungen in einer Vielzahl von Arten, darunter Säugetiere 5-7. Sowohl Umwelt-und genetischen Manipulationen zu Modell DR in C eingesetzt elegans, einschließlich Kultivierung Würmer in einem semi-definierten flüssigen Medium 8,9, Verdünnung der bakteriellen Nahrungsquelle in einem flüssigen Medium 10-12, und die Verwendung von Verhalten...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Carbenicillin </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> In Wasser gelöst. </td></tr><tr><td> Kanamycin </td><td> Roche </td><td> 10106801001 </td><td> In Wasser gelöst. </td></tr><tr><td> Bacto-Pepton </td><td> Becton, Dickinson and Company </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-Trypton </td><td> Becton, Dickinson and Company </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto-Hefeextrakt </td><td> Becton, Dickinson and Company </td><td> DF0127071 </td><td></td></tr><tr><td> Agar </td><td> Becton, Dickinson and Company </td><td> DF00145170 </td><td></td></tr><tr><td> Sterile Kulturröhrchen </td><td> USA Scientific </td><td> 1485-0810 </td><td></td></tr><tr><td> Spektrophotometer </td><td> Amersham Biosciences </td><td> Gene Quant pro </td><td> Amersham ist jetzt Teil von GE Healthcare. </td></tr><tr><td> Präpariermikroskop </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td> Zentrifuge </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Reduktion der Nahrungsaufnahme, ohne Unterernährung oder Hunger ist bekannt, dass Lebensdauer zu erhöhen und das Einsetzen der verschiedenen altersbedingten Erkrankungen in einer Vielzahl von Arten, darunter Säugetiere. Es führt auch zu einer Abnahme des Körpergewichts und der Fruchtbarkeit sowie niedrigere Plasma-Glukose, Insulin und IGF-1 in dieser Tiere. Diese Behandlung wird oft als diätetische Einschränkung (DR) oder kalorische Restriktion (CR) bezeichnet. Der Fadenwurm Caenorhabditis elegans wurde als ein wichtiger Modellorganismus für die Erforschung der Biologie des Alterns entwickelt. Sowohl Umwelt-und genetischen Manipulationen zu Modell DR verwendet worden und haben gezeigt, dass Lebensdauer in C verlängern elegans. Allerdings sind viele der gemeldeten DR Studien in C. elegans wurden durch die Weitergabe von Tieren in flüssigen Medien, während die meisten genetischen Studien in der alternden Feld auf der Standard-festem Agar in Petrischalen durchgeführt wurden durchgeführt. Hier präsentieren wir Ihnen eine DR-Protokoll unter Verwendung von Standard solide NGM-Agar-Basis-Platte mit abgetöteten Bakterien.

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Hier präsentieren wir ein Protokoll für die Durchführung von festen Platte für die Ernährung Einschränkung in<em&gt; C. elegans</em&gt; Mit abgetöteten Bakterien.

Solide Plate-basierte diätetische Einschränkung in Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

solid-plate-based-dietary-restriction-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K Aufrufe.  University of Michigan. Hier präsentieren wir ein Protokoll für die Durchführung von festen Platte für die Ernährung Einschränkung in C. elegans Mit abgetöteten Bakterien.

Serie: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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 ...

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 ...

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, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Nahrungsergänzung von mehrfach ungesättigten Fettsäuren<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantifizierung des gewebespezifischen proteostatischen Rückgangs bei Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Modellierung altersassoziierter neurodegenerativer Erkrankungen bei Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

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 ...