Damos las gracias a Samuel Ward, Diane C. batidos y Gregory Nelson por ser pionero en esta técnica. Damos las gracias a Pavan Kadandale y Geldziler Brian de videos que muestran los espermatozoides activados, los miembros del laboratorio de Singson útil para los debates; CGC para las cepas y los NIH por su apoyo a través de la subvención (R01HD054681).

1) El enriquecimiento de la población masculina <ol><li> Dependiendo de la necesidad de experimentación, un gran número de hombres se puede obtener mediante el empleo de una de las siguientes estrategias: <ol><li> gran población de hombres de tipo salvaje se puede obtener mediante el cruce 5 machos de tipo salvaje y hermafroditas 1 en un pequeño jardín de OP50 cabeza de serie en el centro de la placa de NGM. Aproximadamente el 50% de la generación siguiente serán hombres de tipo salvaje. </li><li> él-5 (e1490) o lo-8 (e1489) hermafroditas lanzar gran cantidad de hombres. lo-5 y lo-8 machos son fértiles y no hay ningún defecto evidente en la morfología de los espermatozoides y función. Por lo tanto, él o ella-5-8 machos pueden ser utilizados en lugar de los hombres de tipo salvaje para muchos experimentos. </li></ol></li></ol> 2) La identificación y el aislamiento de los hombres <ol><li> Examinar la morfología de la cola de los gusanos, la cola del gusano macho es redondeada. </li><li> Elige L4 hombres etapa y la transferencia a un plato NGM sembradas con E. coli OP50 y dejarlas crecer por un día o dos. Cada vez los hombres célibes, en ausencia de hermafroditas evitar la pérdida de los espermatozoides y por lo tanto gran número de espermátidas estaría disponible durante el procedimiento experimental. </li><li> En el día de la disección, la transferencia de los varones célibes sobre una placa de NGM sin bacterias y dejar que se arrastran durante unos minutos. Este paso ayuda a eliminar la pequeña capa de E. coli adheridos en la superficie de los gusanos. Por otra parte, el volumen de la pipeta pequeño de buffer M9 (6 g de Na 2 HPO 4, 3 g KH 2 PO 4, 5 g de NaCl, 0,25 g MgSO 4 .7 H 2 O por litro) en un vidrio de reloj y la transferencia de los hombres en el buffer. </li></ol> 3) La disección de los hombres y en la activación in vitro <ol><li> Tome un portaobjetos de vidrio y marca un pequeño círculo con un bolígrafo de Papanicolaou. Pipeta de 30 a 50 l de medio Sperm 1X (50 mM HEPES, 25 mM KCl, 45 mm de NaCl, 1 mM MgSO 4, 5 mM CaCl 2, 10 mM de dextrosa, pH 7.8) dentro del círculo. El círculo hidrofóbico ayuda a conservar el medio de esperma dentro de sus límites. </li><li> La transferencia de los machos de 5-10 en el medio de los espermatozoides. </li><li> Viendo la diapositiva bajo el microscopio, mantenga unido dos jeringas con agujas de calibre 27 en ambas manos. </li><li> Use una aguja a &#39;mantener&#39; el gusano y el uso de la otra aguja para cortar el extremo posterior de los hombres para liberar espermátidas. </li><li> Cortar el gusano habitualmente expulsa gran cantidad de esperma de forma instantánea. Las espermátides son visibles como minuto &quot;gránulos&quot; bajo el microscopio estereoscópico. Sin embargo, a veces una parte o la mayoría de las espermátides pueden ser retenidos dentro de la carcasa. En ese caso, suavemente &quot;arrastrando&quot; el cadáver sobre el portaobjetos de vidrio, puede facilitar la liberación de esperma de la canal. </li><li> Para activar las espermátides, diseccionar los gusanos en medio de esperma que contiene 1X E Pronasa (20μg/ml) y dejar que repose durante 5 minutos. </li><li> No deje que los portaobjetos se sequen en ningún momento del experimento. Añadir media de esperma más 1X si es necesario. </li></ol> 4) Visualización de spermtids y espermatozoides <ol><li> Con cuidado, coloque un cubreobjetos sobre la superficie de la muestra disecada. </li><li> Encontrar el campo de esperma en aumento bajo (10 veces) </li><li> Los detalles finos de esperma o espermatozoides pueden ser observados por mudarse a un aumento mayor - por lo general en aceite de inmersión 100X. </li></ol> 5) Los resultados representativos Un ejemplo de la imagen DIC de esperma masculino aislado de C. elegans se muestra en la Figura 1. Espermátidas son de forma esférica y los núcleos son prominentes. In vitro de esperma activo se muestran en la Figura 2. <img src="/files/ftp_upload/2336/2336fig1.jpg" alt="Figura 1" /> Figura 1. DIC imagen de C. elegans espermátidas. <img src="/files/ftp_upload/2336/2336fig2.jpg" alt="Figura 2" /> Figura 2. Imagen de DIC in vitro C activada elegans espermatozoides.

<ol>
	<li>Kimble, J., Crittenden, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+germline+stem+cells,+entry+into+meiosis,+and+the+Sperm/Oocyte+decision+in+Caenorhabditis+elegans.">Controls of germline stem cells, entry into meiosis, and the Sperm/Oocyte decision in Caenorhabditis elegans.</a> Annual Review of Cell and Developmental Biology. 23, 405-433 (2007).</li><li>Lhernault, S. W., Roberts, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. , 273-301 (1995).</li><li>Hodgkin, J., Horvitz, H., 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=Nondisjunction+Mutants+of+the+Nematode+Caenorhabditis+elegans.">Nondisjunction Mutants of the Nematode Caenorhabditis elegans.</a> Genetics. 91, 67-94 (1979).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+male+C.+elegans+anatomy.">Introduction to male C. elegans anatomy.</a> WormAtlas. , (2008).</li><li>Shakes, D., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+spermiogenesis+in+C.+elegans:+a+pharmacological+and+genetic+analysis.">Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis.</a> Dev Biol. 134, 189-200 (1989).</li><li>Nelson, G. A., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+fusion,+pseudopod+extension+and+amoeboid+motility+are+induced+in+nematode+spermatids+by+the+ionophore+monensin.">Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin.</a> Cell. 19, 457-464 (1980).</li><li>L'Hernault, S. W. <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+and+cell+biology+of+spermatogenesis+in+the+nematode+C.+elegans.">The genetics and cell biology of spermatogenesis in the nematode C. elegans.</a> Molecular and Cellular Endocrinology. 306, 59-65 (2009).</li><li>Chatterjee, I., Richmond, A., Putiri, E., Shakes, D., Singson, A. <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+spe-38+gene+encodes+a+novel+four-pass+integral+membrane+protein+required+for+sperm+function+at+fertilization.">The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization.</a> Development. 132, 2795-2808 (2005).</li><li>Xu, X., Sternberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+sperm+TRP+protein+required+for+sperm-egg+interactions+during+fertilization.">A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization.</a> Cell. 114, 285-297 (2003).</li><li>Nelson, G., Roberts, T., Ward, S. <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+spermatozoan+locomotion:+amoeboid+movement+with+almost+no+actin.">Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin.</a> J Cell Biol. 92, 121-131 (1982).</li><li>Machaca, K., DeFelice, L. J., L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chloride+channel+localizes+to+Caenorhabditis+elegans+spermatids+and+chloride+channel+blockers+induce+spermatid+differentiation.">A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation.</a> Dev Biol. 176, 1-16 (1996).</li><li>LaMunyon, C., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+viability+of+mutant+and+manipulated+sperm+by+artificial+insemination+of+Caenorhabditis+elegans.">Assessing the viability of mutant and manipulated sperm by artificial insemination of Caenorhabditis elegans.</a> Genetics. 138, 689-692 (1994).</li></ol>

Además de analizar mutantes Espe, este protocolo tiene otras aplicaciones importantes, como el análisis de la morfología del espermatozoide con el envejecimiento. Espermátides y espermatozoides aislados utilizando este protocolo puede ser utilizado en otros experimentos posteriores, tales como, la inmunotinción de tipo salvaje y el espermatozoide mutante 8, 9, motilidad de los espermatozoides en las diapositivas 10, las mediciones fisiológicas 9, 11, o incluso la inseminación arti...

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<td>Tuberculin syringe</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>309623</td>
<td>Syringe: 1 ml, needle: 27G X &frac12; in</td>
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<td>Pap pen</td>
<td>&nbsp;</td>
<td>Zymed</td>
<td>00-8888</td>
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<td>VWR VistaVision HistoBond Microscope Slides</td>
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isolation and in vitro activation of caenorhabditis elegans sperm

Los varones y hermafroditas son las dos formas sexuales se encuentran naturalmente en el nematodo C. elegans. Los espermatozoides son ameboides producida tanto por hombres y hermafroditas. En la primera fase de la gametogénesis, las células germinales de los hermafroditas se diferencian en cantidad limitada de espermatozoides - alrededor de 300 - y se almacenan en una pequeña &quot;bolsa&quot; llama la espermateca. Más tarde, los hermafroditas producen continuamente ovocitos 1. Por el contrario, los machos producen exclusivamente espermatozoides durante toda su vida adulta. Los machos producen espermatozoides tanto que representa&gt; 50% del total de células en un típico gusano de dos adultos. Por lo tanto, aislar los espermatozoides de los machos es más fácil que de la de los hermafroditas. Sólo una pequeña proporción de los hombres son naturalmente generado debido a la espontánea no disyunción del cromosoma X 3. Cruzando hermafroditas con los hombres o, más convenientemente, la introducción de mutaciones que dan lugar a él (alta incidencia de varones) fenotipo son algunas de las estrategias a través del cual se puede enriquecer a la población masculina 3. Los machos pueden distinguirse fácilmente de los hermafroditas mediante la observación de la morfología de la cola 4. La cola es hermafrodita en punta, mientras que la cola masculina se completa con las estructuras de apareamiento. Corte de la cola libera gran cantidad de esperma almacenado en el interior del aparato reproductor masculino. La disección se realiza bajo un microscopio estéreo con agujas de calibre 27. Desde espermátidas no están físicamente conectados con cualquier otra célula, la presión hidráulica expulsa contenido interno del cuerpo masculino, incluyendo dos espermátidas. Los machos son directamente disecados en una pequeña gota de &quot;Medio de esperma&quot;. Espermátidas son sensibles a la alteración en el pH. Por lo tanto, HEPES, un compuesto con una buena capacidad de amortiguación se utiliza en los medios de esperma. La glucosa y otras sales presentes en los medios de comunicación ayudan a mantener el esperma presión osmótica para mantener la integridad de los espermatozoides. Post-meiótica diferenciación de las espermátidas en espermatozoides se denomina espermiogénesis o la activación de los espermatozoides. Batidos 5, 6 y Nelson mostraron con anterioridad que espermátidas redondas pueden ser inducidas a diferenciarse en espermatozoides mediante la adición de diferentes compuestos como la activación de pronasa E. Aquí se demuestra in vitro espermiogénesis de C. elegans espermátidas con pronasa E. Espermiogénesis éxito es pre-requisito para la fertilidad y por lo tanto, los mutantes defectuosos en la espermiogénesis son estériles. Hasta ahora, varios mutantes han demostrado que son defectuosos específicamente en el proceso de espermiogénesis 7. Anomalía encontrada durante la activación in vitro de la novela de SPE (espermatogénesis defectuosa) mutantes que nos ayuden a descubrir más jugadores que participan en este evento.

Watch this Scientific Journal Video about Isolation and In vitro Activation of Caenorhabditis elegans Sperm at JoVE.com

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Un protocolo para el aislamiento y la activación de las espermátidas de varón<em&gt; C. elegans</em&gt; Se describe aquí. Cortar el extremo posterior de esperma macho libera. Las espermátides se puede activar mediante la adición de la proteasa.

El aislamiento y la In vitro La activación de Caenorhabditis elegans El esperma

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

isolation-and-in-vitro-activation-of-caenorhabditis-elegans-sperm

Research

JoVE Journal

Biology

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

16.2K vistas.  Rutgers University. Un protocolo para el aislamiento y la activación de las espermátidas de varón C. elegans Se describe aquí. Cortar el extremo posterior de esperma macho libera. Las espermátides se puede activar mediante la adición de la proteasa.

Video: Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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 range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Placa Sólida basado en la restricción dietética 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 ...

Investigación

Biología

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.

Time-lapse microscopía de embriogénesis temprana en 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 ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Vampiric Aislamiento de líquido extracelular de<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos de sincronización y de observación

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.

Extracción y análisis de prostaglandina en<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.

La suplementación dietética de ácidos grasos poliinsaturados en<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, ...

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.

El cultivo de<em&gt; Caenorhabditis elegans</em&gt; En tres dimensiones en el Laboratorio

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Nucleótido y extracciones de proteínas de Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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.

Medición de las tasas de consumo de oxígeno en intacto Caenorhabditis elegans

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

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.

Purificación y análisis de las vesículas extracelulares de Caenorhabditis elegans

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