Este trabajo fue apoyado por una Glenn / AFAR avances en la concesión de Gerontología y Subvenciones del NIH 1R01AG031108-01 a MKGS es apoyado por el NIH formación conceder P30AG013280. MK es una Ellison Medical Foundation de Nueva Académico en el envejecimiento.

Parte 1: Preparar nematodo crecimiento de los medios (NGM) placas En esta sección se describe la preparación de los platos NGM sólida para su uso en el experimento de vida. Un experimento de vida útil básica requiere dos tipos de placas: las placas estándar NGM, que no contienen aditivos, y las placas Amp / FUDR, que tienen la ampicilina (Amp) y fluorodesoxiuridina (FUDR) añadido a la NGM. La ampicilina se utiliza para prevenir la contaminación bacteriana externa. FUDR inhibe la división celular, reduce la producción de huevos, y evita que los huevos de incubar. El uso de FUDR para el análisis de la longevidad no afecta vida adulta y elimina la necesidad de transferir los gusanos cada pocos días con el fin de separarlos de la larva en crecimiento. Ambos tipos de placas se siembran con E. coli OP50 bacterias, que posteriormente asesinado por la exposición a los rayos UV. <ol><li> Prepare NGM (véase la Sección 5 de la receta y notas de almacenamiento) y placas de identificación de Petri. Usted tendrá una placa de Petri de 60 mm por cada 10 ml de NGM. Si usted está comenzando con NGM previamente preparados sólidos, continúe con el paso 1.2. Si usted está comenzando con NGM recién autoclave, vaya al paso 1.4. </li><li> Fundir completamente NGM sólida en el microondas en alto en 30 pulsos de segundo. Los medios de comunicación remolino entre los pulsos para evitar burbujas de gas a presión se acumule. </li><li> NGM en lugar de líquido 55 ° C baño de agua o en el banco que se enfríe. </li><li> Una vez NGM alcanza los 55 ° C añadir 33 l de FUDR 150 mm y 100 L de 100 mg / ml de ampicilina por cada 100 ml NGM (por Amp / FUDR placas) y agitar para mezclar. Para placas NGM sin medicamentos adicionales vaya directamente al paso 1.5. </li><li> Utilizando una técnica estéril, pipeta de 10 mL en cada NGM 60 mm placa de Petri. Evitar la formación de burbujas, si es posible. Si se forman burbujas, que se pueden hacer estallar con una punta de la pipeta. </li><li> Deja los platos en la mesa con tapa para permitir que el NGM para solidificar y secar. Si es posible, dejar las placas en el banco durante 2 días antes de la adición de bacterias, pero un día va a funcionar si las placas son necesarios antes. </li><li> El día antes de terminar de secar los platos, líquido de semilla cultura LB con una colonia de una racha dulce de E. coli OP50 bacterias en agar LB. Usted debe preparar por lo menos 1 ml de una noche a la mañana OP50 cultura por placa de 60 mm. </li><li> Lugar OP50 cultura en 37 ° C agitador y permitir que las bacterias crecen durante la noche (la cultura debe llegar a la saturación). </li><li> Pellet OP50 bacterias al girar a 3.500 g durante 10 minutos. </li><li> Eliminar el 90% de las bacterias sobrenadante y resuspender a concentrar la cultura de 10x. </li><li> Pipetear 100 L de 10x concentrada OP50 la cultura en el centro de las placas NGM solidificado. Placas de agitar suavemente para extender cultivo de bacterias, si es necesario (lo ideal bacteria debe cubrir el área central de la NGM sin llegar cerca de las paredes de placas). Trate de no tocar la punta de la pipeta a la superficie de la NGM, como defectos en la superficie de los medios de comunicación permiten que las lombrices se introducen en la NGM. </li><li> Deja los platos en la mesa durante la noche para permitir que la cultura bacterias se seque en la NGM sólida (por lo general tarda aproximadamente 24 horas). </li><li> Una vez seco, exponer la superficie de las placas a una dosis UV suficiente para detener el crecimiento de las bacterias. Si estás utilizando un Stratalinker UV Stratagene 2400: <ul><li> Coloque las placas en la Stratalinker y quite las tapas </li><li> Cierre la puerta y encender el Stratalinker </li><li> &quot;Energía&quot; de prensa </li><li> Introduzca &#39;9999 &#39;usando el teclado </li><li> &quot;Inicio&quot; de prensa </li><li> Las placas estarán expuestas a los rayos UV durante unos 5 minutos </li></ul></li><li> Las placas con las bacterias muertas por UV se pueden almacenar al revés a 4 ° C durante un máximo de un mes. </li></ol> Parte 2: Realizar un huevo de tiempo por la que se adquieren a una edad sincronizado población de animales En esta sección se genera una población de gusanos con una escotilla común-la fecha. Esto se logra al permitir que los adultos reproductivamente activas para poner los huevos en un plato por un período definido de tiempo y permitiendo que los huevos para desarrollarse. <ol><li> (Opcional) Transferencia de aproximadamente 20 gusanos adultos jóvenes a una placa de NGM fresco sin FUDR. Deja las placas a 25 ° C a fin de que los gusanos se propaguen y que comer a través de todo el alimento de bacterias. Después de la comida bacteriana que se ha consumido gusanos recién nacidos entrará en la etapa de crecimiento arrestado Dauer. Usted empezará a ver larva Dauer todo dentro de una semana, dependiendo de cómo muchos gusanos que transfiera a la placa inicial. Dauer larvas se pueden utilizar para los próximos pasos para un máximo de un mes. </li><li> (Opcional) Transferencia de 20 a 30 Dauer larva de una placa de NGM fresco sin FUDR. En presencia de alimentos larva Dauer se convertirán en adultos reproductivamente activas dentro de 2 días y siguen siendo reproductivo activos por unos pocos días después a 25 ° C. </li><li> Transferencia de 10 a 15 adultos reproductivamente activos a una placa de NGM fresco sin FUDR. Este plato se conoce como la puesta de huevos tiempo (TEL) de placas. </li><li> Deje la placa de TEL a 25 ° C durante 6 horas a fin de que tque el tiempo los gusanos para poner sus huevos. </li><li> Eliminar los gusanos adultos de la placa TEL. Placa puede ser inspeccionada visualmente para detectar los huevos antes de retirar los adultos. Si los gusanos no han establecido un número suficiente de huevos de la placa de TEL se puede dejar a 25 ° C hasta un total de 24 horas antes de la eliminación de los gusanos adultos. </li><li> Coloque la placa de TEL a 20 ° C hasta que los huevos han eclosionado y los gusanos se han desarrollado hasta la etapa larval L4 (esto por lo general, toma 2 días de tipo silvestre de C. elegans, pero puede tomar más tiempo para las cepas con fenotipos de desarrollo lento). </li></ol> Parte 3: Puntuación de los animales vida En esta sección seguimos la edad sincronizado población de gusanos en la parte 2 hasta que mueren. Los gusanos se mantienen en Amp / FUDR placas para evitar que la producción de huevos y la contaminación bacteriana y se considera muerto cuando no responden a los estímulos externos. <ol><li> Transferencia L4 larva de semillas Amp / FUDR placas. Para cada cepa o afección que se estudia, es típico para configurar 2-3 placas con 25 a 30 gusanos por placa. Imágenes de la C. etapas de la vida elegans se proporcionan para los de referencia (Figura 1) 1. </li><li> Lugar Amp / FUDR placas a 20 ° C durante 24 horas. </li><li> Después de 24 horas, visualmente evaluar los gusanos, los medios de comunicación, y las bacterias. Transferencia de gusanos a un nuevo amplificador / FUDR placas si alguno de los siguientes se observa: <ul><li> Los gusanos se han comido la mayoría de los alimentos bacteriana. A principios de los gusanos experimento duración de la vida tendrá que ser transferido 1 a 2 veces a la semana para evitar que las bacterias están agotando. </li><li> Un número significativo de larvas presentes. Esto es generalmente un indicio de que los gusanos fueron trasladados a las placas Amp / FUDR como adultos jóvenes en lugar de L4s y fueron capaces de poner unos huevos antes de la FUDR entró en vigor. El FUDR suele prevenir las larvas de llegar a ser adultos completa, pero de vez en cuando unos pocos se convierten en adultos y se confunden con los animales de experimentación. </li><li> Bacterias vivas / cada vez se observan. En general, la combinación de amplificador y UV-matar se asegurará de que ninguna bacteria viva contaminar el experimento, pero a veces ocurre. </li><li> El crecimiento de hongos que se observa en los medios de comunicación. Si se detecta a tiempo, el crecimiento de hongos por lo general se puede cortar con una punta de la pipeta o una espátula. Una vez que crece hasta ser más grande que unos pocos milímetros de diámetro, por lo general es más fácil de transferir a los gusanos a una nueva placa. </li><li> Utilizar las posibilidades que la disección, determinar si cada gusano está vivo o muerto. </li><li> Golpear suavemente la placa. El gusano está vivo si se mueve en respuesta a la interceptación. </li><li> Si el gusano de ellos no responden a tocar la placa, zoom en la región de la cabeza. </li><li> Golpee suavemente la cabeza del gusano con el pico de platino de transferencia. Puntuación del gusano como muerta si no responde moviendo su cabeza. </li><li> Gusanos muertos pueden ser retirados de la placa. </li></ul></li><li> Anote la fecha y el número de gusanos que están vivos y los muertos. </li><li> Volver placas a 20 ° C. </li><li> Repita los pasos 3.3 a 3.6 cada 2 ó 3 días hasta que todos los gusanos han muerto. </li></ol> Parte 4: Los resultados representativos. Los datos en bruto producido por un experimento de nematodos vida es una lista de fechas con los números correspondientes de los gusanos que están vivos y los muertos de cada cepa ensayada. El número de gusanos que se mueren cada día suele ser invertido para calcular la proporción de gusanos vivos en todos los días (Figura 2A), que se representa gráficamente como una curva de supervivencia (Figura 2B, el día de la puesta de huevos tiempo se considera el día 0 ). La esperanza de vida para cada individuo del gusano en el estudio se puede calcular a partir del recuento de los gusanos que mueren cada día y se utiliza para el cálculo de tiempo de vida media y la mediana para la comparación entre las cepas. El recuento del número de gusanos vivos en cada día no se utiliza directamente en el análisis de vida útil. Gusanos mantenidos en medios sólidos de vez en cuando &#39;huir&#39;, o se arrastran hacia arriba la pared de la placa o por debajo de la media. El número de gusanos vivos en cada día se puede utilizar para determinar cómo los gusanos, muchos huyeron a lo largo del experimento. Gusanos que huyen normalmente se extirpan de análisis. Como punto de referencia, la duración de vida típica de la mediana de N2, el C. elegans cepa de tipo salvaje, mantenidos en el UV-mató a las bacterias a 20 ° C es de aproximadamente 25 días como medida de huevo. Parte 5: Soluciones <table border="0"><tbody><tr><td colspan="2"> Nematodo de crecimiento de los medios (NGM), 100 ml: </td></tr><tr><td colspan="2"> Combine: </td></tr><tr><td> 0,3 g </td><td> NaCl </td></tr><tr><td> 0,25 g </td><td> Peptona </td></tr><tr><td> 2 g </td><td> Agar </td></tr><tr><td colspan="2"> Autoclave durante 40 minutos y dejar enfriar a 55 ° C, a continuación, añadir: </td></tr><tr><td> 100 L </td><td> 1 M MgSO 4 </td></tr><tr><td> 100 L </td><td> 5 mg / ml de colesterol </td></tr><tr><td> 100 L </td><td> 1 M de CaCl2 </td></tr><tr><td> 1.625 ml </td><td> 1,5 M pH 6,0 KPI </td></tr><tr><td colspan="2"> NGM líquido puede ser utilizado de inmediato para verter en las placas o se deja solidificar y se almacenan a largo plazo a temperatura ambiente </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> Caldo Luria (LB), 1 litro: </td></tr><tr><td> 10 g </td><td> BactoTryptone </td></tr><tr><td> 5 g </td><td> Extracto de levadura </td></tr><tr><td> 10 g </td><td> NaCl </td></tr><tr><td> 10 ml </td><td> 1 M Tris pH 8,0 </td></tr><tr><td> 1 L </td><td> agua desionizada </td></tr><tr><td colspan="2"> Autoclave y almacenar a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M MgSO 4, 300 ml: </td></tr><tr><td> 73,95 g </td><td> MgSO4 </td></tr><tr><td> 300 ml </td><td> agua desionizada </td></tr><tr><td colspan="2"> Autoclave y almacenar a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 5 mg / ml de colesterol, 200 ml: </td></tr><tr><td> 1 g </td><td> colesterol </td></tr><tr><td> 200 ml </td><td> 100% de etanol </td></tr><tr><td colspan="2"> Filtro de esterilizar y almacenar a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M CaCl 2, 500 ml: </td></tr><tr><td> 27,75 g </td><td> CaCl2 </td></tr><tr><td> 500 ml </td><td> agua desionizada </td></tr><tr><td colspan="2"> Filtro de esterilizar y almacenar a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1,5 M pH 6,0 KPI, 1L: </td></tr><tr><td colspan="2"> Combine: </td></tr><tr><td> 31,4 g </td><td> KPI dibásico </td></tr><tr><td> 179,6 g </td><td> KPI monobásico </td></tr><tr><td> 850 ml </td><td> agua desionizada </td></tr><tr><td colspan="2"> Calor mientras se mezcla para permitir KPI en disolverse. Ajustar el pH a 6.0 con NaOH 10 N. </td></tr><tr><td colspan="2"> Añadir agua desionizada hasta 1 L. </td></tr><tr><td colspan="2"> Autoclave y almacenar a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M Tris, pH 8,0: </td></tr><tr><td> 60,57 g </td><td> Tris </td></tr><tr><td> 400 ml </td><td> agua desionizada </td></tr><tr><td colspan="2"> Ajustar el pH a 8.0 con HCl. </td></tr><tr><td colspan="2"> Añadir agua desionizada hasta 500 ml. </td></tr><tr><td colspan="2"> Filtro de esterilizar y almacenar a temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 150 mM fluorodesoxiuridina (FUDR), 10 ml: </td></tr><tr><td> 0,3693 g </td><td> FUDR </td></tr><tr><td> 10 ml </td><td> agua destilada estéril </td></tr><tr><td colspan="2"> Almacenar a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 100 mg / ml de ampicilina (Amp), 10 ml: </td></tr><tr><td> 1 g </td><td> Ampicilina </td></tr><tr><td> 10 ml </td><td> agua destilada estéril </td></tr><tr><td colspan="2"> Almacenar a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 50 mg / ml carbenicilina (CARB), 10 ml: </td></tr><tr><td> 500 mg </td><td> Carbenicilina </td></tr><tr><td> 10 ml </td><td> agua destilada estéril </td></tr><tr><td colspan="2"> Almacenar a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M isopropílico β-D-tiogalactopiranósido (IPTG), 10 ml: </td></tr><tr><td> 2,38 g </td><td> IPTG </td></tr><tr><td> 10 ml </td><td> agua destilada estéril </td></tr><tr><td colspan="2"> Almacenar a -20 ° C. </td></tr></tbody></table><img src="/files/ftp_upload/1152/Figure%201.png" alt="la figura 1" /> Figura 1. Imágenes de campo claro de la C. elegans etapas de la vida, incluyendo huevos, de los cuatro estadios larvales (L1 - L4) y adulto. Todos los paneles muestran hermafroditas, excepto la esquina inferior derecha, que muestra a un hombre adulto (Imagen de madera (1988)). <img src="/files/ftp_upload/1152/Figure%202.png" alt="la figura 2" /> Figura 2. Los resultados representativos de una C. la vida elegans experimento lapso comparar salvaje N2 tipo de cepa a cepa que contiene una mutación en el gen daf-2. (A) Una tabla que muestra los datos recogidos, incluyendo el número de días desde que los gusanos fueron los huevos, el número de gusanos muertos observados cada día, y el porcentaje de la muestra original que permanecen vivos en cada día (calculada a partir de los recuentos diarios de los gusanos muertos) . (B) Supervivencia curvas correspondientes a los datos de vida útil previsto en (A). <imgsrc = &quot;files/ftp_upload/1152/Figure / 3.jpg&quot; alt = &quot;figura 3&quot; /&gt; Figura 3. Comparación Representante de la lipofuscina entre adultos jóvenes y viejos C. elegans. Imágenes brillantes de campo se muestran a la izquierda y DAPI imágenes del canal de fluorescencia a la derecha. Los paneles superiores muestran un gusano de 4 días de edad y paneles inferiores muestran un gusano de 11 días de edad (medida a partir de huevo).

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El control genético de la longevidad se ha estudiado extensamente en C. elegans, en gran parte debido a la facilidad y rapidez con que puede ser tiempo de vida determinado. El protocolo discutido en este artículo se describe un marco básico para la obtención de datos reproducibles esperanza de vida en C. elegans y también se puede aplicar a las especies relatednematode. 2 Al hacer algunos cambios sencillos estos procedimientos pueden ser adaptados para medir duración de la vida bajo un...

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<td>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0145-17-0 (214530)</td>
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<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
<tr>
<td>BactoTryptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0123-17-3 (211705)</td>
<td>&nbsp;</td>
<tr>
<td>BactoPeptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0118-17-0 (211677)</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9699248 (1332-01)</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>C0109</td>
<td>&nbsp;</td>
<tr>
<td>Cholesterol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C75209</td>
<td>&nbsp;</td>
<tr>
<td>Fluorodeoxyuridine (FUDR)</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl &#x03B2;-d-Thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>I2481C5</td>
<td>&nbsp;</td>
<tr>
<td>KPi Dibasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087862 (3252-01)</td>
<td>&nbsp;</td>
<tr>
<td>KPi Monobasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087861 (3246-01)</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9561800 (2500-01)</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S251</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0886-17-0 (288620)</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring caenorhabditis elegans life span on solid media

El envejecimiento es un proceso degenerativo caracterizado por un deterioro progresivo de los componentes celulares y orgánulos que resulta en la mortalidad. El nematodo<em&gt; Caenorhabditis elegans</em&gt; Se ha convertido en un modelo principal utilizado para estudiar la biología del envejecimiento. Debido a que prácticamente todos los subsistemas biológico sufre deterioro funcional que aumenta la edad, duración de la vida es el objetivo primario de interés cuando se considera la tasa total de envejecimiento. En los nematodos, esperanza de vida se define normalmente como el número de días que un animal sigue siendo sensible a los estímulos externos. Los nematodos pueden propagarse, ya sea en medios líquidos o en el medio sólido en placas, y se han desarrollado técnicas para medir la duración de la vida en ambas condiciones. Aquí se presenta un protocolo generalizado para medir la duración de la vida de los nematodos mantienen en medios sólidos nematodo crecimiento y alimentados con una dieta de muertos UV-bacterias. Estos procedimientos se pueden adaptar fácilmente para abarcar ensayo de la vida bajo diversas condiciones comunes, incluyendo una dieta a base de bacterias vivas, la restricción dietética, y la interferencia de ARN.

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span on Solid Media at JoVE.com

Measuring Caenorhabditis elegans Life Span on Solid Media

En este artículo se presenta un protocolo general para medir la duración de la vida de los nematodos mantienen en medios sólidos con UV-muertos alimentaria bacteriana.

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 ...

measuring-caenorhabditis-elegans-life-span-on-solid-media

Research

JoVE Journal

Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

29.7K vistas.  University of Washington. En este artículo se presenta un protocolo general para medir la duración de la vida de los nematodos mantienen en medios sólidos con UV-muertos alimentaria bacteriana.

Video: Measuring Caenorhabditis elegans Life Span on Solid Media

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

Cuantificar cronológico levadura Duración de la vida por consecuencia de células envejecidas

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Investigación

Biología

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Medición de Span replicativa vida en la levadura en ciernes

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

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 ...

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 ...

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 ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

El cultivo<em&gt; Caenorhabditis elegans</em &gt; En axénico Liquid Media y Creación de transgénicos Worms por bombardeo de micropartículas

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

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

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

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 ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Uso de Caenorhabditis elegans para detectar interacciones de chaperonas específicas del tejido

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Imágenes Libres De Etiquetas De La Dinámica De Almacenamiento De Lípidos En Caenorhabditis elegans Usando Microscopía De Dispersión Raman Estimulada

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

Detección de alto rendimiento de aislados microbianos con impacto en la salud de Caenorhabditis elegans

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...