Este trabajo fue apoyado por un premio de piloto del Centro de choque Nathan (P30 AG13283) y una subvención R01 (R01 AG028516) del Instituto Nacional sobre el Envejecimiento (NIA) para A.-LH

<html> 1. El establecimiento de una curva de calibración para la estimación de las concentraciones de bacterias En esta sección se describe un método para la estimación de la concentración de bacterias de cualquier cepa determinada bacteria (por ejemplo, OP50, HT115) que se utilizará como fuente de alimento para los experimentos de restricción dietética en C. elegans. Las curvas de calibración independientes deben ser establecidos para cada cepa de la bacteria utilizada. <ol><li> Inocular una colonia de E. coli OP50 bacterias seleccionar de una racha fresca de bacterias en agar LB en 3 ml de caldo LB líquido. </li><li> Lugar OP50 cultura en 37 ° C agitador y permitir que las bacterias crecen durante la noche. </li><li> Prepare serie 2 diluciones de la suspensión bacteriana de la cultura durante la noche (2x, 4x, 8x, 16x, 32x). </li><li> Medir la absorbancia (densidad óptica) de cada dilución a 600 nm por espectrofotometría por triplicado. </li><li> Volver a diluir cada suspensión bacteriana 1.000.000 veces (1000 veces la dilución en dos ocasiones). </li><li> Propagación 0,4 ml de suspensión bacteriana de cada dilución de manera uniforme sobre una placa de agar LB 100 mm. </li><li> Colocar placas de LB con OP50 en una incubadora a 37 ° C durante 18-24 horas. </li><li> Cuente el número de colonias de bacterias cultivadas en cada plato. El recuento de colonias se usan para calcular UFC (unidades formadoras de colonias) por ml para cada muestra. </li><li> La relación entre la medida OD 600 valores y OP50 concentraciones de bacterias puede ser establecida por el trazado de OD 600 en función de la concentración de bacterias (ufc / ml) y se realiza un análisis de regresión lineal (Figura 1). </li></ol> Soluciones: <table border="1"><tbody><tr><td colspan="2"> Caldo Luria (LB), 1 litro: </td></tr><tr><td > 10 g </td><td> Bacto-triptona </td></tr><tr><td> 5 g </td><td> Extracto de levadura </td></tr><tr><td> 10 g </td><td> NaCl </td></tr></tbody></table> Añadir agua desionizada a 1L. Autoclave y almacenar a temperatura ambiente. 2. Preparación de la placa sólida basada en la dieta de restricción de giro (DEG), las placas En esta sección se describe la preparación de platos para su uso en base placa sólida-la dieta de restricción de giro (DEG) los experimentos. Normalmente, se considera una concentración de bacterias de 1 x 10 11 UFC / mL como la condición de la alimentación ad libitum y 1 x 10 8 UFC / mL como la condición de una alimentación óptima DR para este método. Sin embargo, las concentraciones de 4-6 diferentes bacterias pueden ser necesarios para establecer una completa relación dosis-dependiente de DEG y sus respuestas. <ol><li> Inocular una colonia de E. Coli OP50 bacterias en 3 ml de caldo LB líquido. </li><li> Lugar de la cultura OP50 en 37 ° C agitador y permitir que las bacterias crezcan durante 6-8 horas. </li><li> La transferencia de la cultura OP50 a un nuevo frasco contiene 500 ml de caldo de LB, y el lugar de la cultura OP50 de vuelta en 37 º C durante la noche coctelera. </li><li> Mida el diámetro exterior de 600 de la cultura OP50 para determinar la concentración de bacterias (la concentración se calcula utilizando la curva de calibración generada en la parte 1). Diluciones adecuadas puede ser necesario para una medición precisa de la absorción. </li><li> Pellet OP50 bacterias por centrifugación a 1.500 g durante 20 minutos. </li><li> Volver a suspender las bacterias a una concentración de 1 x 10 12 UFC / ml. </li><li> Preparar 10 mL cultivo de bacterias en seis diferentes concentraciones: 1 x 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 ufc / ml. </li><li> Pipetear 0,2 ml de cultivo de bacterias en el centro de una placa de 60 mm solidificado NGM. Evite tocar la superficie de la placa con la punta de la pipeta, como rasguños en la superficie permite a los gusanos se introducen en las placas de agar. </li><li> Coloque las placas en incubadora a 37 ° C durante 1 hora. </li><li> Aplicar 10μL de 100mg/ml carbenicilina y 10 l de kanamicina 50mg/ml a cada placa para detener el crecimiento de las bacterias. </li><li> Tienda placas con diferentes concentraciones de los antibióticos bacterias muertas a 4 ° C para su uso futuro (hasta un mes). Es importante preparar y almacenar las placas suficiente para que el experimento planeado todo por lo menos 1-2 días por venir. Utilizando el mismo lote de placas para los experimentos de todo es preferible. </li><li> Para verificar la viabilidad de las bacterias, sacar algunas bacterias de las placas de DEG y la extendió sobre una placa de NGM vacío con carbenicilina y kanamicina. Colocar la placa en incubadora a 37 ° C durante 6-8 horas y luego verificar el crecimiento de las bacterias (no el crecimiento de bacterias debe ser observado). </li></ol> Soluciones <table border="1"><tbody><tr><td colspan="2"> Nematodo de crecimiento de los medios (NGM), 1 litro: </td></tr><tr><td > 3 g </td><td> NaCl </td></tr><tr><td> 2,5 g </td><td> Bacto-peptona </td></tr><tr><td> 20g </td><td> Agar </td></tr></tbody></table>ove_content &quot;Autoclave&gt; durante 40 minutos y dejar enfriar a 65 ° C, a continuación, añadir: <table border="1"><tbody><tr><td > 1 mL </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 mL </td><td> 1 M de CaCl2 </td></tr><tr><td> 1 mL </td><td> 5 mg / ml de colesterol </td></tr><tr><td> 25 ml </td><td> Fosfato de Potasio 1 M, pH 6,0 </td></tr></tbody></table> Inmediatamente vierta líquido en NGM 60 mm x 15 mm placas de Petri en alícuotas de 10 mL 3. La creación de experimentos DEG Para la mayoría de los estudios relacionados con el DEG, como el análisis de duración de la vida y perfiles de la fertilidad, la preparación de una edad sincronizado población de gusanos es esencial. Para otros estudios, sólo la transferencia de los gusanos de una placa normal OP50 a una placa de DEG para iniciar la restricción dietética. En esta sección, un protocolo para establecer un análisis de ciclo de vida para los animales de DEG se describe como un ejemplo. <ol><li> Utilizar un selector de gusano de platino para la transferencia de 20 a 30 adultos reproductivamente activos en varias placas NGM regular con OP50. Dependiendo de la cantidad de huevos necesarios para los experimentos, los más adultos pueden ser necesarias. </li><li> Dejar las placas a 20 ° C durante 4 horas para permitir la puesta de huevos. </li><li> Retire los adultos de la placa. Las placas deben ser inspeccionados visualmente para los huevos. </li><li> Dejar las placas a 20 ° C y permitir que la progenie de convertirse en la etapa adulta Late-L4/day 1. Por lo general, este es de 3 días para que los gusanos N2 tipos silvestres a 20 ° C. </li><li> La transferencia de un número adecuado de adultos el día 1 en la placa de DEG para iniciar la restricción dietética. </li><li> Para un análisis de ciclo de vida, creó un total de 6-8 con placas 10-20 gusanos por placa para cada condición de DEG. </li><li> Puntuación de la viabilidad de cada gusano cada 2 días hasta que todos los gusanos han muerto. Es fundamental que los gusanos se transfirieron a placas de DEG fresca cada 12-48 horas para evitar la privación completa de alimentos. </li></ol> 4. Los resultados representativos: Se muestra en la Figura 1 es un gráfico de una curva de calibración representante para la estimación de la concentración OP50 en relación con OD 600. La ecuación generada por el análisis de línea de regresión puede utilizarse para todos los cálculos futuros de OP50 concentraciones de bacterias. Muestra en la Figura 2 es un ejemplo de la determinación del efecto de los tratamientos crónicos en DEG significa vida útil de las cepas de gusanos diferentes. La línea azul representa una normal dependiente de la dosis respuesta de tipo salvaje gusanos de DEG. La línea roja indica que el efecto de la vida útil de DEG es parcialmente suprimida por la RRD-2 oe, mientras que el daf-16 (mu86) no tiene ningún efecto sobre la SDR-inducida por prolongación de la vida. Información sobre métodos para realizar análisis de ciclo de vida de C. elegans se describe en la literatura previa 1-3. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Figura 1" /> Figura 1. Representante resultado de una curva de calibración para la estimación de la E. Coli OP50 concentración. OP50 concentración se representa como una función de medida OD 600 valores. Las barras de error = SD cultivo bacteriano noche a la mañana se diluyó 1.5x, 2x, 4x, 8x, 16x, 32x antes de la medición de absorbancia, y posteriormente diluido 1.000 x dos veces antes de la medición ufc. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Figura 2" /> Figura 2. Representante resultado de una C. elegans experimento esperanza de vida comparando los animales salvajes N2 tipo (azul) con dos diferentes mutantes (verde y rojo) en respuesta a los DEG (la figura de Ching et al. 4). Las barras de error = 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subfield+history:+caloric+restriction,+slowing+aging,+and+extending+life.">Subfield history: caloric restriction, slowing aging, and extending life.</a> Sci Aging Knowledge Environ. , RE2-RE2 (2003).</li><li>Masoro, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+caloric+restriction+and+ageing.">Overview of caloric restriction and ageing.</a> Mech Ageing Dev. 126, 913-922 (2005).</li><li>Houthoofd, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenic+growth+up-regulates+mass-specific+metabolic+rate,+stress+resistance,+and+extends+life+span+in+Caenorhabditis+elegans.">Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span in Caenorhabditis elegans.</a> Exp Gerontol. 37, 1371-1378 (2002).</li><li>Houthoofd, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+reduction+of+metabolic+rate+in+food+restricted+Caenorhabditis+elegans.">No reduction of metabolic rate in food restricted Caenorhabditis elegans.</a> Exp Gerontol. 37, 1359-1369 (2002).</li><li>Klass, M. 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La restricción dietética (DR) se sabe que aumenta la vida útil y retrasar la aparición de diversas enfermedades relacionadas con la edad en una amplia gama de especies, incluyendo mamíferos 7.5. Manipulaciones ambientales y genéticos se han utilizado para el modelo DR en C. elegans, incluidos los gusanos de cultivo en un medio líquido semi-definido 8,9, la dilución de la fuente de alimento de bacterias en un medio líquido 10 a 12, y el uso de mutantes de comportamiento ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Carbenicilina </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> Disuelto en el agua. </td></tr><tr><td> Kanamicina </td><td> Roche </td><td> 10106801001 </td><td> Disuelto en el agua. </td></tr><tr><td> Bacto-peptona </td><td> Becton, Dickinson and Company </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-triptona </td><td> Becton, Dickinson and Company </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto-extracto de levadura </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> Tubo de cultivo estéril </td><td> EE.UU. Científico </td><td> 1485-0810 </td><td></td></tr><tr><td> Espectrofotómetro </td><td> Amersham Biosciences </td><td> Gene Quant pro </td><td> Amersham ahora es parte de GE Healthcare. </td></tr><tr><td> Microscopio de disección </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td> Centrífugo </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Reducción de la ingesta de alimentos sin desnutrición o inanición se sabe que aumenta la vida útil y retrasar la aparición de diversas enfermedades relacionadas con la edad en una amplia gama de especies, incluyendo mamíferos. También provoca una disminución en el peso corporal y la fertilidad, así como niveles más bajos de glucosa en plasma, la insulina y el IGF-1 en estos animales. Este tratamiento se refiere a menudo como la restricción dietética (DR) o la restricción calórica (CR). El nematodo Caenorhabditis elegans se ha convertido en un importante organismo modelo para estudiar la biología del envejecimiento. Manipulaciones ambientales y genéticos se han utilizado para el modelo DR y han demostrado prolongar la vida de C. elegans. Sin embargo, muchos de los estudios reportados en la República Dominicana C. elegans se realizaron mediante la propagación de los animales en un medio líquido, mientras que la mayoría de los estudios de genética en el campo del envejecimiento se realiza en el estándar de agar sólido en placas de Petri. Aquí se presenta un protocolo estándar de DR con NGM agar sólido basado en la placa con bacterias muertas.

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

Aquí se presenta un protocolo para la realización de placa sólida basada en la restricción dietética en<em&gt; C. elegans</em&gt; Con bacterias muertas.

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 ...

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

Research

JoVE Journal

Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K vistas.  University of Michigan. Aquí se presenta un protocolo para la realización de placa sólida basada en la restricción dietética en C. elegans Con bacterias muertas.

Video: 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.

Medición Caenorhabditis elegans Duración de la vida en el medio sólido

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

Investigación

Biología

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.

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 ...

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 ...

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 ...

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 ...

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.

Cuantificación de la disminución proteostática específica del tejido en Caenorhabditis elegans

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

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.

Automatización de la cuantificación agregada en 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 ...