Este trabalho foi financiado por um prêmio de piloto do Centro de Choque Nathan (P30 AG13283) e uma subvenção R01 (R01 AG028516) do Instituto Nacional do Envelhecimento (NIA) para A.-LH

<html> 1. Estabelecer uma curva de calibração para a estimativa das concentrações de bactérias Esta seção descreve um método para a estimativa da concentração de bactérias por qualquer estirpe de bactérias dada (por exemplo, OP50, HT115) que será usado como fonte de alimento para os experimentos A restrição alimentar em C. elegans. Curvas de calibração em separado devem ser estabelecidos para cada bactéria cepa utilizada. <ol><li> Inocular uma única colônia de E. coli OP50 bactérias escolhidos de uma raia fresca de bactérias em agar LB em 3 mL de caldo LB líquido. </li><li> Cultura local OP50 em 37 ° C shaker e permitir que as bactérias cresçam durante a noite. </li><li> Prepare serial 2 diluições da suspensão bacteriana a partir da cultura durante a noite (2x, 4x, 8x, 16x, 32x). </li><li> Medir a absorbância (densidade óptica) de cada diluição em 600 nm em espectrofotômetro em triplicado. </li><li> Dilua cada suspensão bacteriana 1000000 vezes (1000 vezes diluição duas vezes). </li><li> Spread 0,4 mL de suspensão bacteriana de cada diluição uniformemente sobre uma placa de agar 100 milímetros LB. </li><li> Coloque placas de LB com OP50 em uma incubadora de 37 ° C por 18-24 horas. </li><li> Contar o número de colônias de bactérias cultivadas em cada prato. A contagem de colônias são usados ​​para calcular ufc (unidades formadoras de colônia) por ml para cada amostra. </li><li> A relação entre a medida OD 600 valores e OP50 concentrações de bactérias pode ser estabelecida através da representação gráfica OD 600 como uma função da concentração de bactérias (UFC / mL) e realizando uma análise de regressão linear (Figura 1). </li></ol> Soluções: <table border="1"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td > 10 g </td><td> Bacto-triptona </td></tr><tr><td> 5 g </td><td> Extrato de levedura </td></tr><tr><td> 10 g </td><td> NaCl </td></tr></tbody></table> Adicione água deionizada para 1L. Autoclave e armazenar em temperatura ambiente. 2. Preparação de sólidos placa baseada na dieta de restrição (SDR) placas Esta seção descreve a preparação de placas para uso em chapas sólidas baseada em dieta de restrição (SDR) experimentos. Normalmente, considera-se uma concentração de bactérias de 1 x 10 11 ufc / mL como o ad libitum condição de alimentação e 1 x 10 8 ufc / mL como a condição ideal de alimentação DR para este método. No entanto, 4-6 diferentes concentrações de bactérias pode ser necessário para estabelecer uma relação dose-dependente completa para SDR e suas respostas. <ol><li> Inocular uma única colônia de E. OP50 coli bactéria em 3 mL de caldo LB líquido. </li><li> Coloque a cultura OP50 em 37 ° C shaker e permitir que as bactérias cresçam por 6-8 horas. </li><li> Transferir a cultura OP50 para um balão novo contendo 500 mL de caldo LB, e coloque a cultura OP50 de volta em 37 ° C shaker durante a noite. </li><li> Medir o OD 600 da cultura OP50 para determinar a concentração de bactérias (a concentração é calculado utilizando a curva de calibração gerada em Part 1). Diluições apropriadas podem ser necessários para uma medição precisa da absorbância. </li><li> Pellet OP50 bactérias por centrifugação a 1.500 g por 20 minutos. </li><li> Ressuspender as bactérias a uma concentração de 1 x 10 12 ufc / mL. </li><li> Preparar 10 mL cultura de bactérias em seis diferentes concentrações: 1 x 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 ufc / mL. </li><li> Pipetar 0,2 mL de cultura de bactérias para o centro de uma placa de 60 mm solidificou NGM. Evite tocar na superfície da placa com a ponta da pipeta, como nicks na superfície permitem worms para penetrar na placas de ágar. </li><li> Placas lugar em 37 ° C incubadora por 1 hora. </li><li> Aplicar 10μL de 100mg/ml carbenicilina e 10 mL de canamicina 50mg/ml a cada placa para prender o crescimento das bactérias. </li><li> Placas de loja com diferentes concentrações de antibióticos de bactérias mortas a 4 ° C para uso futuro (até um mês). É importante preparar e armazenar placas suficiente para todo o experimento planejado pelo menos 1-2 dias adiante. Usando o mesmo lote de placas para todo o experimento é preferível. </li><li> Para verificar a viabilidade das bactérias, raspar algumas bactérias para fora das placas DSE e espalhá-lo em uma placa NGM vazio com carbenicilina e canamicina. Coloque a placa em 37 ° C incubadora de 6-8 horas e em seguida, verificar para o crescimento das bactérias (sem o crescimento de bactérias deve ser observado). </li></ol> Soluções <table border="1"><tbody><tr><td colspan="2"> Nematóide Crescimento Media (NGM), 1L: </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;&gt; Autoclave por 40 minutos e deixe esfriar até 65 ° C, em seguida, acrescentar: <table border="1"><tbody><tr><td > 1 mL </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 mL </td><td> 1 M CaCl 2 </td></tr><tr><td> 1 mL </td><td> Colesterol 5 mg / mL </td></tr><tr><td> 25 mL </td><td> Fosfato de Potássio 1M, pH 6,0 </td></tr></tbody></table> Imediatamente despeje NGM líquido sobre 60 milímetros x 15 milímetros pratos petri em 10 alíquotas mL 3. Criação de experimentos SDR Para a maioria dos estudos SDR-relacionados, tais como análise de tempo de vida e perfil de fertilidade, a preparação de uma era sincronizada população de vermes é essencial. Para outros estudos, simplesmente transferir worms de uma placa de OP50 regular para uma placa de SDR para iniciar a restrição alimentar. Nesta seção, um protocolo para criar uma análise de ciclo de vida para os animais SDR é descrito como um exemplo. <ol><li> Use um seletor de verme de platina para transferir 20-30 adultos reprodutivamente ativos em várias placas NGM regular com OP50. Dependendo do número de ovos necessários para os experimentos, mais adultos podem ser necessárias. </li><li> Deixar as placas a 20 ° C por 4 horas para permitir a postura de ovos. </li><li> Remove os adultos a partir da placa. As placas devem ser inspeccionados visualmente para os ovos. </li><li> Deixar as placas a 20 ° C e permitir que a progênie de desenvolver na fase adulta Late-L4/day 1. Normalmente, isso leva de 3 dias para worms N2 selvagens tipos a 20 ° C. </li><li> Transferência de um número adequado de adultos dia 1 na placa de SDR para iniciar a restrição alimentar. </li><li> Para uma análise vida, criou um total de 6-8 com 10-20 placas worms por placa para cada condição SDR. </li><li> Pontuação a viabilidade de cada minhoca a cada 2 dias até que todos os vermes já morreram. É fundamental que worms ser transferidos para placas DSE fresco todos os 12-48 horas para evitar a privação completa de alimentos. </li></ol> 4. Resultados representativos: Mostrado na Figura 1 é um gráfico de uma curva de calibração representante para a estimativa das OP50 concentração em relação ao OD 600. A equação gerada pela análise de regressão liner pode ser usado para todos os cálculos futuro da OP50 concentrações de bactérias. Mostrado na Figura 2 é um exemplo de doseamento do efeito dos tratamentos SDR crônica na vida útil média de cepas de vírus diferentes. A linha azul representa uma resposta dose-dependente normal de tipo selvagem worms para SDR. A linha vermelha indica que o efeito tempo de vida de SDR é parcialmente suprimida pela DRR-2 oe, enquanto daf-16 (mu86) não tem efeito sobre SDR-induzida extensão vida. Método de detalhes para a realização de análise de ciclo de vida em C. elegans é descrita na literatura anterior 1-3. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Figura 1" /> Figura 1. Resultado Representante de uma curva de calibração para a estimativa de E. Concentração de Escherichia OP50. OP50 concentração é plotado como uma função da medida OD 600 valores. Barras de erro = SD cultura Overnight bacteriana foi diluída 8x 1.5x, 2x, 4x, 16x, 32x antes da medição de absorbância, e posteriormente diluído 1.000 x duas vezes antes da medição ufc. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Figura 2" /> Figura 2. Representante resultado de um C. elegans experimento vida comparando animais tipo selvagem N2 (azul) com dois mutantes diferentes (verde e vermelho) em resposta a SDR (Figura de Ching et al. 4). Barras de erro = 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech Ageing Dev. 6, 413-429 (1977).</li><li>Bishop, N. A., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+neurons+mediate+diet-restriction-induced+longevity+in+C.+elegans.">Two neurons mediate diet-restriction-induced longevity in C. elegans.</a> Nature. 447, 545-549 (2007).</li><li>Panowski, S. H., Wolff, S., Aguilaniu, H., Durieux, J., Dillin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PHA-4/Foxa+mediates+diet-restriction-induced+longevity+of+C.+elegans.">PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans.</a> Nature. 447, 550-555 (2007).</li><li>Lakowski, B., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+caloric+restriction+in+Caenorhabditis+elegans.">The genetics of caloric restriction in Caenorhabditis elegans.</a> Proc Natl Acad Sci U S A. 95, 13091-13096 (1998).</li><li>Kaeberlein, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+extension+in+Caenorhabditis+elegans+by+complete+removal+of+food.">Lifespan extension in Caenorhabditis elegans by complete removal of food.</a> Aging Cell. 5, 487-494 (2006).</li><li>Greer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+AMPK-FOXO+pathway+mediates+longevity+induced+by+a+novel+method+of+dietary+restriction+in+C.+elegans.">An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans.</a> Curr Biol. 17, 1646-1656 (2007).</li></ol>

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Restrição dietética (DR) é conhecido por aumentar a longevidade ea retardar o aparecimento de várias doenças relacionadas à idade em uma ampla gama de espécies, incluindo mamíferos 5-7. Manipulações ambientais e genéticos têm sido usados ​​para modelar DR em C. elegans, incluindo worms cultura em um meio semi-definido líquido 8,9, diluição da fonte de alimento de bactérias em meio líquido 10-12, e uso de mutantes comportamento que se alimentam em um ritmo m...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Carbenicilina </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> Dissolvido em água. </td></tr><tr><td> Canamicina </td><td> Roche </td><td> 10106801001 </td><td> Dissolvido em água. </td></tr><tr><td> Bacto-peptona </td><td> Becton, Dickinson e companhia </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto-triptona </td><td> Becton, Dickinson e companhia </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto levedura-extrato de </td><td> Becton, Dickinson e companhia </td><td> DF0127071 </td><td></td></tr><tr><td> Agar </td><td> Becton, Dickinson e companhia </td><td> DF00145170 </td><td></td></tr><tr><td> Tubo de cultura estéril </td><td> EUA Scientific </td><td> 1485-0810 </td><td></td></tr><tr><td> Espectrofotômetro </td><td> Amersham Biosciences </td><td> Gene Quant pro </td><td> Amersham agora faz parte da GE Healthcare. </td></tr><tr><td> Microscópio de dissecação </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td> Centrifugador </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Redução da ingestão alimentar sem desnutrição ou fome é conhecido por aumentar a longevidade ea retardar o aparecimento de várias doenças relacionadas à idade em uma ampla gama de espécies, incluindo mamíferos. Ela também provoca uma diminuição no peso corporal e fertilidade, bem como níveis mais baixos de glicose plasmática, insulina e IGF-1 nesses animais. Este tratamento é muitas vezes referida como restrição dietética (DR) ou a restrição calórica (CR). O nemátodo Caenorhabditis elegans tem emergido como um organismo modelo importante para o estudo da biologia do envelhecimento. Manipulações ambientais e genéticos têm sido usados ​​para modelar DR e têm mostrado que prolonga a vida em C. elegans. No entanto, muitos dos estudos relatados DR em C. elegans foram feitas por animais propagação em meios líquidos, enquanto a maioria dos estudos genéticos na área do envelhecimento foram feitos no ágar padrão sólido em placas de petri. Aqui apresentamos um protocolo DR usando o padrão sólido NGM placa de ágar-base com as bactérias mortas.

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

Aqui apresentamos um protocolo para a realização de restrição placa baseada em sólidos na dieta<em&gt; C. elegans</em&gt; Com bactérias mortas.

Placa sólida baseada na restrição 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 ...

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Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K visualizações.  University of Michigan. Aqui apresentamos um protocolo para a realização de restrição placa baseada em sólidos na dieta C. elegans Com bactérias mortas.

Vídeo: 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.

Medição Caenorhabditis elegans Expectativa de Vida em meios sólidos

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

Biologia

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.

Isolamento e In vitro A ativação do 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 ...

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.

Lapso de tempo Microscopia de embriogênese inicial em 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: Sincronização e observação

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.

Extração de prostaglandina e Análise em<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.

A suplementação dietética de ácidos graxos poliinsaturados em<em&gt; Caenorhabditis elegans</em

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Medição das taxas de consumo de oxigênio em intacta 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.

Quantificando o declínio proteostático específico do tecido em elegans caenorhabditis

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

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

Modelagem de Doenças Neurodegenerativas Associadas à Idade em Caenorhabditis elegans

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

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Automatizando quantificação agregada em 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 ...