Reconocemos el Centro de Genética Caenorhabditis para C. cepas elegans. El apoyo financiero fue proporcionado por el Instituto de Investigación de Fox Terry. También reconocemos el apoyo otorgado a EP de la Rolande y Marcel Gosselin Graduate Beca y la beca de formación CIHR / FRSQ en FRN53888 investigación del cáncer del Programa de Formación de Investigación del Cáncer de McGill Integrado.

1. Preparación de los reactivos <ol><li> Preparación de los medios de comunicación para C. crecimiento elegans (en este caso, los animales de tipo salvaje y los animales FLCN-1 (ok975) mutantes). <ol><li> Prepárese Modificado Sólo Bacto-peptona (MYOB) mezcla seca de Youngren que contiene 5,5 g de Tris HCl, 2,4 g de base Tris, 31 g de Bactopetone, 20 g de NaCl y 0,08 g de colesterol. Mezclar bien con agitación. NOTA: Esta mezcla es suficiente para preparar 10 litros de medio MYOB. NOTA: placas de medio de crecimiento normal (NGM) podrían utilizarse en lugar de placas de 27 MYOB. Sin embargo, el mismo tipo de placas se debe utilizar para comparaciones válidas entre las cepas y entre repeticiones independientes. En este protocolo, sólo utilizamos placas MYOB. </li><li> Preparar 1 l de medio MYOB añadiendo 6 g de MYOB mezcla seca, 17 g de agar y completar hasta 1 l con H2O y autoclave durante 45 minutos a 122 ° C. Verter en las placas y dejar secar a temperatura ambiente durante 2 días. </li><li> Utilizando una técnica estéril, inocular 100 ml de culture de medio de caldo LB con E. bacterias coli OP50 y crecen O / N a 37 ° C. </li><li> Placas MYOB Semilla con E. bacterias coli OP50. Deje que crezca a temperatura ambiente durante 48 horas. </li></ol></li><li> Preparación para la regulación a la baja de genes utilizando RNAi <ol><li> Preparar platos RNAi alimentación MYOB. Proceda como en los pasos 1.1.1 y 1.1.2. Después de la esterilización en autoclave, deje que el medio se enfríe a aproximadamente 55 ° C y añadir 1 ml de 1 M isopropil β-D-1-tiogalactopiranósido (IPTG) y 1 ml de ampicilina 100 mM. </li><li> Streak E. coli HT115 bacterias transformadas con un plásmido, que expresa ya sea de control o FLCN EV-1 RNAi específica sobre ampicilina (50 mg / ml) / tetraciclina (15 g / ml) en placas de agar. Crecer cultivo O / N a 37 ° C. </li><li> Escoja colonias e inocular E. coli HT115 bacterias transformadas con un plásmido que expresa ya sea de control o FLCN EV-1 RNAi específica y crecen en cultivo LB / ampicilina (50 mg / ml) a medio y O / N a 37 &amp; #176; C. </li><li> Placas de semillas con E. coli HT115 EV bacterias o HT115 FLCN-1 RNAi bacterias. NOTA: Mantenga las placas de 48 horas a temperatura ambiente antes de su uso. Si RNAi desmontables por la alimentación es en efectivo, utilice otros métodos de entrega de la ds-RNA 28. </li></ol></li><li> La inducción de estrés oxidativo usando paraquat (PQ): <ol><li> Preparar tampón M9 mediante la mezcla de 3 g de KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl y 0,25 g de MgSO 4 · 7 H 2 O. Llevar hasta 1 L con agua destilada. Autoclave solución durante 45 minutos a 122 ° C botellas y almacenar a temperatura ambiente. </li><li> En el día del ensayo, preparar la solución de M9 / PQ-que contiene. Para este protocolo, utilizar mM PQ 100 (0.1028 g de PQ disuelto en 4 ml de tampón M9 llena toda una placas de 96 pocillos con 40 l de M9 / PQ por pocillo). PRECAUCIÓN: PQ es un compuesto peligroso y altamente tóxico. Manipular de acuerdo con las directrices apropiadas. NOTA: Cuando se ejecuta primero un nuevo stren o una nueva condición, se recomienda analizar la supervivencia en concentraciones crecientes de PQ para determinar la mejor concentración para utilizar con el fin de matar a los animales dentro de 7 a 10 h. </li></ol></li></ol> 2. Preparación de sincronizada-Edad L4 Población <ol><li> Transferencia 20 adultos hermafroditas grávidas a una placa MYOB fresco sembrado con E. bacterias coli OP50. Preparar varias placas basadas en el número de gusanos necesarios durante el ensayo. </li><li> Deje que se ponen huevos durante 6 horas y luego utilizando un gusano alambre de platino pick de quitar las madres. Compruebe las placas bajo el microscopio para evaluar el número de huevos puestos. </li><li> Permita que los huevos eclosionan y crecen a 20 ° C durante 48 horas. En este momento, los animales están en un estado adulto finales L4 / joven. Escoja mismos animales de escenario y la transferencia a los pozos que contienen solución-PQ. NOTA: Desde 48 horas de incubación sólo se aplica a los mutantes que crecen de manera similar a los animales de tipo salvaje, es importante vigilar el desarrollotasa de mutantes recién probado para asegurar que se ajusta a la tasa de desarrollo de tipo salvaje, de lo contrario, deben utilizarse otras líneas de tiempo. </li><li> Alternativamente, utilizar técnicas de sincronización. Generar población sincronizada de L1 gusanos larvas utilizando una solución de hipoclorito (25 ml de agua, 125 ml de 5,25% de hipoclorito de sodio, 50 ml de 4 M de NaOH; envuelva botella con papel de aluminio para aislar de la luz). Sin embargo, para este ensayo, no se recomienda, ya intensa blanqueo podría afectar el comportamiento de los animales durante el ensayo. PRECAUCIÓN: Manipular solución de hipoclorito de acuerdo con las directrices. NOTA: Al usar el ARNi para regular por el gen de interés, sincronizar los hermafroditas como se describe en la sección 2, con la excepción de la transferencia de las madres a las placas de RNAi sembradas con la bacteria específica RNAi. Cuando la caída con el ARNi es débil, se recomienda la alimentación para varias generaciones. </li></ol> 3. Realizar el estrés oxidativo Resistencia Ensayo &lt;/ P&gt; <ol><li> Pipetear 40 l de la solución 100 mM PQ (preparado fresco) a cada pocillo de una placa de 96 pocillos. Utilice al menos 12 pozos como repeticiones de cada condición (tratamiento, mutante, etc.). El uso de un gusano selección alambre de platino, transferir 5-8 animales larvas L4 a cada bien indicando la hora de inicio y la hora de finalización. </li><li> Al iniciar otra condición, tenga en cuenta el tiempo de inicio y la hora de finalización. Colocar la placa a 20 ° C en el tiempo de incubación entre la transferencia de los gusanos y anotando animales muertos una hora después de la hora de inicio. </li><li> Utilizando el microscopio de disección, cuente la supervivencia cada hora. Agite suavemente la placa antes de comenzar a contar. Indique gusanos que no se mueven, incluso después de ver una luz de alta intensidad, como animales muertos. Asimismo, indique el número de animales vivos. NOTA: En cuanto a la forma de gusano, las colas y el movimiento de la cabeza en la alta ampliación, ayudar a determinar gusanos muertos de vida. </li><li> Repita este paso cada hora hasta que la mayoría de los gusanos están muertos. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Determinación del porcentaje de supervivencia en Every Time Point <ol><li> Calcular el número total de animales por condición sumando el número total de animales transferidos a cada pocillo. No haga caso de los animales que se encuentren dañados o muertos tras la transferencia. </li><li> Por cada punto de tiempo, calcular el número total de animales muertos por enfermedad (suma de los animales muertos en los 12 pozos). Por cada punto de tiempo, calcular el porcentaje de mortalidad (número de animales muertos dividido por el número total de animales y multiplicado por 100) y porcentaje de supervivencia (100 menos ciento muerte). </li><li> Repita este ensayo de al menos 3 veces independientes. </li></ol>

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The authors declare that they have no competing financial interests.

C. elegans es un organismo modelo atractivo para estudiar la resistencia al estrés oxidativo genéticamente in vivo, ya que puede ser fácilmente cultivada, y rápidamente conduce a un gran número de descendencia genéticamente idéntica. Múltiples métodos para medir la resistencia al estrés oxidativo se han descrito anteriormente y se basan en la administración de suplementos de placas de cultivo con varias fuentes de ROS tales como PQ, rotenona, H 2 O 2, y juglone 25,...

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

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

En eucariotas, la fosforilación oxidativa que tiene lugar en la cadena de transporte de electrones de la mitocondria es el principal motor de la producción de energía en forma de ATP. Especies reactivas del oxígeno (ROS) son un subproducto natural de este proceso. A pesar de su importante papel como moléculas de señalización, excesiva ROS puede conducir a daños en el ADN, carbonilación de proteínas, y la oxidación de los lípidos. Un desequilibrio entre la producción y la desintoxicación ROS causa el estrés oxidativo, que conduce a agotamiento de la energía, el daño celular, y desencadena la muerte celular 1,2. El estrés oxidativo contribuye al envejecimiento y al desarrollo de muchas enfermedades que amenazan la vida, incluyendo cáncer, diabetes, enfermedades cardiovasculares y neurodegenerativas 3-9. Las células han desarrollado estrategias de defensa enzimáticos y no enzimáticos para mantener los niveles de ROS adecuadas y para proteger a sus electores contra el daño oxidativo 1,2. La superóxido dismutasa (SOD) enzimas actúan primero en convert superóxido a H 2 O 2, que más tarde se convierte en el agua por la catalasa o enzimas peroxidasa. Estrategias para no enzimáticos de defensa incluyen principalmente moléculas que reaccionan más rápidamente con ROS en comparación con macromoléculas celulares, protegiendo los componentes celulares esenciales. A pesar de la función protectora de ROS desintoxicación enzimas, algunas moléculas ROS escapan los mecanismos de defensa antioxidante y conducen al daño oxidativo. Detección, reparación, y la degradación de los componentes celulares dañados son estrategias esenciales de defensa durante el estrés oxidativo 1,2. Vías de señalización implicadas en la resistencia a la tensión y el estrés oxidativo en concreto son altamente conservadas evolutivamente 10,11. A diferencia de los experimentos de cultivo de células, donde las condiciones organismal son sólo parcialmente reproducidos, el estudio del estrés oxidativo en organismos modelo 12,13 tiene un gran significado. C. elegans es un nematodo de vida libre que puede ser fácil y económicamente culrado en un césped de bacterias en medios de agar. Es de tamaño pequeño (alrededor de 1 mm de longitud) y normalmente crece como un hermafrodita auto-fertilización, lo que facilita las manipulaciones genéticas. Tiene un ciclo de vida rápido y una alta capacidad reproductiva, produciendo aproximadamente 300 crías por generación, por lo que es una poderosa herramienta para realizar cribados genéticos a gran escala 14. El C. elegans genoma está completamente secuenciado y el 40-50% de los genes se predice para ser homólogos de genes asociados a enfermedades humanas 15-18. La caída de los genes de interés utilizando RNAi es rápida y fácil en C. elegans. génica por la regulación se puede lograr mediante la alimentación de animales de la E. bacterias coli que albergan un plásmido que expresa el ARN de doble cadena que se dirige el mRNA de interés 19. Por lo tanto, la determinación de la función de genes utilizando pantallas de RNAi gran escala tiene un gran impacto en la comprensión de las enfermedades humanas como el cáncer 20,21. Estudios de oresistencia al estrés xidative en C. elegans han conducido a la identificación de conservados mecanismos de resistencia al estrés oxidativo 13,22. Algunas vías identificadas son vías comunes que modulan la longevidad y la resistencia a otros factores de estrés, así como la hipoxia, el calor, y el estrés osmótico. Estas vías incluyen la señalización de la insulina, la señalización de TOR y la autofagia. Otras vías importantes implican la desintoxicación de ROS como las enzimas superóxido dismutasa y catalasa enzimas, o en la reparación de daños tales como choque térmico y chaperón proteínas 11,13,22. Este protocolo describe cómo determinar la resistencia al estrés oxidativo de C. elegans en líquido. Utilizamos FLCN-1 (ok975) y de tipo salvaje animales para demostrar el protocolo ya que hemos demostrado anteriormente una mayor resistencia al estrés oxidativo sobre la pérdida de FLCN-1 (ok975) en C. elegans 23. También hemos demostrado que este aumento de la resistencia dependeen AMPK y la autofagia, un eje de señalización que mejora la bioenergética celular y promueve la resistencia al estrés 23. PQ es un factor de estrés oxidativo que interfiere con la cadena de transporte de electrones para producir especies de oxígeno reactivo 24. El mismo ensayo se podría adaptar y otras fuentes de ROS o compuestos que generan ROS podría ser utilizado como H 2 O 2 y rotenona. Ensayos similares se han desarrollado en las placas donde se utilizan bajas concentraciones de PQ 25,26. La ventaja de este ensayo es que es muy rápido, y los resultados se podría obtener en un día. Además, el volumen total de líquido usado para llevar a cabo el ensayo de la resistencia al estrés oxidativo en placas de 96 pocillos es baja en comparación con el volumen utilizado para preparar placas PQ-que contienen. Por lo tanto, la cantidad de PQ es utilizado en el ensayo de líquido es bajo, lo que hace que el ensayo de bajo costo y limita la producción de desechos tóxicos. Sin embargo, las limitaciones de este ensayo en comparación con los ensayos de placa incluyen el lack de comida en el ensayo de líquido y la menor concentración de oxígeno en el líquido en comparación con el aire. Estos son factores importantes que en algunos casos, podrían influir en los resultados. Por lo tanto, lo que confirma la reproducibilidad utilizando otros métodos de la resistencia al estrés oxidativo se recomienda para apoyar los resultados obtenidos en este ensayo.

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

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

El estrés oxidativo, que es el resultado de un desequilibrio entre la producción y la desintoxicación de especies reactivas del oxígeno, es un importante contribuyente a trastornos humanos crónicas, incluyendo enfermedades cardiovasculares y neurodegenerativas, diabetes, envejecimiento, y cáncer. Por lo tanto, es importante para estudiar el estrés oxidativo no sólo en sistemas celulares, sino también el uso de organismos enteros. C. elegans es un organismo modelo atractivo para estudiar la genética de las vías de transducción de señales de estrés oxidativo, que se conservan altamente evolutivamente. Aquí, ofrecemos un protocolo para medir la resistencia al estrés oxidativo en C. elegans en líquido. Reactivos Brevemente, ROS inductores como el paraquat (PQ) y H 2 O 2 se disuelven en tampón M9, y las soluciones se dividen en partes alícuotas en los pocillos de una placa de microtitulación de 96 pocillos. L4 Sincronizada / joven C. elegans animales se transfieren a los pocillos (5-8 animales / pocillo) y se mide la supervivenciacada hora hasta que la mayoría de los gusanos están muertos. Cuando se realiza un ensayo de resistencia al estrés oxidativo usando una baja concentración de factores de estrés en las placas, el envejecimiento podría influir en el comportamiento de los animales sobre el estrés oxidativo, lo que podría dar lugar a una interpretación errónea de los datos. Sin embargo, en el ensayo descrito en el presente documento, este problema es poco probable que ocurra ya que se están utilizando sólo L4 / animales adultos jóvenes. Por otra parte, este protocolo es de bajo costo y los resultados se obtienen en un día, lo que hace que esta técnica atractiva para pantallas genéticos. En general, esto le ayudará a entender las vías de transducción de señales de estrés oxidativo, lo que podría traducirse en una mejor caracterización de los trastornos humanos con el estrés oxidativo asociado.

La comparación de tipo salvaje C. elegans para FLCN-1 (ok975) animales mutantes Aquí hemos utilizado PQ 100 mM para determinar la resistencia de tipo salvaje C. elegans animales en comparación con FLCN-1 (ok975) que se ha demostrado para resistir el estrés oxidativo, el calor y la anoxia 23. Después de 4 horas de tratamiento, el 48,3% de los de tipo salvaje sobrevivido en comparación con el 77,8% de supervivencia en-1 FLCN (ok975)...

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

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

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

Medición de la resistencia de estrés oxidativo<em&gt; Caenorhabditis elegans</em&gt; 96 pocillos en placas de microtitulación

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

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

Research

JoVE Journal

Biology

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

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

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

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 ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

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

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

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

Medición Caenorhabditis elegans Duración de la vida en 96 placas de microtitulación Bueno

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

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 ...

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

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

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

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

Capa de hielo: Un método para el cultivo Arabidopsis Y plantas de tomate en las placas de 96 pocillos de alto rendimiento para genotipado

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

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, ...

Analysis of Oxidative Stress in Zebrafish Embryos

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

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

Análisis de estrés oxidativo en embriones de pez cebra

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

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 ...