Este trabalho foi financiado por um Avanços Glenn / AFAR em Gerontologia e Grant Award NIH 1R01AG031108-01 a MKGS é suportado pelo NIH treinamento conceder P30AG013280. MK é um Ellison Medical Foundation Scholar novo no Envelhecimento.

Parte 1: Prepare nematóide crescimento media (NGM) placas Esta seção descreve a preparação das placas NGM sólido para uso no experimento vida. A experiência de extensão básicas da vida requer dois tipos de placas: placas NGM padrão, que não contêm aditivos, e Amp / FUDR placas, que têm ampicilina (Amp) e fluorodeoxyuridine (FUDR) adicionado ao NGM. Ampicilina é usado para prevenir a contaminação bacteriana estrangeiros. FUDR inibe a divisão celular, reduz a produção de ovos, e impede que os ovos de incubação. O uso de FUDR para a análise da longevidade não afeta expectativa de vida de adultos e elimina a necessidade de transferência de worms cada poucos dias, a fim de separá-los de larva a crescer. Ambos os tipos de placas são semeados com E. coli OP50 bactérias, que é posteriormente morto por exposição a UV. <ol><li> Prepare NGM (ver secção 5 para receita e notas de armazenamento) e placas de petri rótulo. Você vai precisar de uma placa de petri 60 milímetros por 10 mL de NGM. Se você está começando com previamente preparado NGM sólida, avance para o passo 1.2. Se você está começando com NGM recém autoclavado, pule para o passo 1.4. </li><li> Derreter completamente NGM sólido por microondas em alta em 30 pulsos segundo. Media redemoinho entre os pulsos para evitar bolhas de gás pressurizado de edificação. </li><li> NGM lugar líquido em 55 ° C ou banho de água na bancada para esfriar. </li><li> Uma vez que NGM atinge 55 ° C adicionar 33 mL de 150 FUDR mM e 100 mL de 100 mg / mL por 100 mL Ampicilina NGM (para Amp / FUDR placas) e agite para misturar. Para placas NGM sem drogas adicionais prosseguir diretamente para o passo 1.5. </li><li> Utilizando uma técnica estéril, pipetar 10 mL em cada placa NGM petri 60 mm. Evite a formação de bolhas, se possível. Se as bolhas se formam, elas podem ser estalado usando uma ponta da pipeta. </li><li> Deixar as placas sobre a bancada com tampas para permitir NGM para solidificar e seco. Se possível, deixe as placas no banco por 2 dias antes da adição de bactérias, mas um dia vai funcionar se as placas são necessários mais cedo. </li><li> No dia antes de terminar a secagem das placas, a semente da cultura LB líquido com uma única colônia a partir de uma raia fresca de E. coli OP50 bactérias em agar LB. Você deve preparar, pelo menos, 1 mL de uma cultura OP50 noite por 60 placa mm. </li><li> Cultura local OP50 em 37 ° C shaker e permitir que as bactérias cresçam overnight (a cultura deve atingir a saturação). </li><li> Pellet OP50 bactérias girando a 3.500 g por 10 minutos. </li><li> Remover 90% das bactérias sobrenadante e ressuspender concentrar cultura 10x. </li><li> Pipetar 100 mL da cultura 10x OP50 concentrada no centro de placas NGM solidificado. Placas agite cuidadosamente para espalhar cultura de bactérias se necessário (o ideal bactérias deve cobrir a área central da NGM sem chegar perto das muralhas da placa). Tente evitar tocar a ponta da pipeta na superfície da NGM, como falhas na superfície dos meios de comunicação permitirá que as minhocas burrow na NGM. </li><li> Deixe placas no banco durante a noite para permitir cultura de bactérias para secar na NGM sólido (normalmente demora cerca de 24 horas). </li><li> Depois de seco, expor a superfície das placas a uma dose UV suficiente para deter o crescimento das bactérias. Se você estiver usando uma Stratalinker UV Stratagene 2400: <ul><li> Placas lugar no Stratalinker e remover as tampas </li><li> Feche a porta e ligar o Stratalinker </li><li> &quot;Energia&quot; de imprensa </li><li> Digite &#39;9999 &#39;usando o teclado </li><li> &#39;Start&#39; imprensa </li><li> As placas serão expostos aos raios UV por aproximadamente 5 minutos </li></ul></li><li> Placas com as bactérias UV-morto pode ser armazenado de cabeça para baixo a 4 ° C por até 1 mês. </li></ol> Parte 2: Executar um ovo cronometrados que estabelece uma idade para a aquisição sincronizada população de animais Nesta seção, vamos gerar uma população de vermes com uma data comum hatch. Isto é conseguido, permitindo que adultos reprodutivamente ativos para colocar ovos em um prato por um período definido de tempo e permitindo que os ovos se desenvolvam. <ol><li> (Opcional) Transferência de aproximadamente 20 vermes adultos jovens a uma placa NGM fresca sem FUDR. Deixar as placas a 25 ° C, a fim de permitir que worms se propaguem e comer através de todos os alimentos bacteriana. Depois que o alimento foi consumido bacteriana worms recém-nascidos vai entrar no estágio de crescimento preso Dauer. Você começará a ver larva Dauer em torno de uma semana, dependendo de quantos worms você transferir para a placa inicialmente. Dauer larva pode ser usado para as próximas etapas para até um mês. </li><li> (Opcional) Transferência larva Dauer 20-30 a uma placa NGM fresca sem FUDR. Na presença de larva de alimentos se tornarão adultos Dauer reprodutivamente ativa dentro de 2 dias e manter-se reprodutivamente ativa por alguns dias depois a 25 ° C. </li><li> Transferência de 10-15 adultos reprodutivamente ativa para uma placa NGM fresca sem FUDR. Esta placa é referida como a postura de ovos cronometrado (TEL) da placa. </li><li> Deixe a placa TEL a 25 ° C durante 6 horas, a fim de permitir que tele worms tempo para botar ovos. </li><li> Remover vermes adultos de placa TEL. Placa pode ser inspeccionados visualmente para os ovos antes de remover os adultos. Se os vermes não tenham estabelecido um número suficiente de ovos a placa TEL podem ser deixados a 25 ° C por até um total de 24 horas antes de retirar vermes adultos. </li><li> Colocar a placa TEL a 20 ° C até que os ovos eclodiram e os vermes desenvolveram ao estágio larval L4 (o que normalmente leva 2 dias para tipo selvagem C. elegans, mas pode demorar mais tempo para as cepas com fenótipos de desenvolvimento lento). </li></ol> Parte 3: animais Partituras de vida Nesta seção, siga as idade sincronizado população de vermes na parte 2 até morrerem. Worms são mantidos em Amp / FUDR placas para impedir a produção de ovos ea contaminação bacteriana e são considerados mortos quando não conseguem responder a estímulos externos. <ol><li> Transferência de larva L4 para semeado Amp / FUDR placas. Para cada estirpe ou condição a ser testada, é típico para configurar 2-3 placas com 25-30 worms por placa. Imagens da C. fases da vida elegans são fornecidos para a referência (Figura 1) 1. </li><li> Lugar Amp / FUDR placas a 20 ° C por 24 horas. </li><li> Após 24 horas, visualmente avaliar worms, mídia e bactérias. Transferência de worms para fresco Amp / FUDR placas se qualquer um dos seguintes é observado: <ul><li> Worms ter comido a maior parte dos alimentos bacteriana. No início da vida worms experimento extensão terá de ser transferida 1-2 vezes por semana para evitar que as bactérias fossem esgotados. </li><li> Um número significativo de larvas estão presentes. Isso geralmente é uma indicação de que os vermes foram transferidos para as placas Amp / FUDR como jovens adultos, em vez de L4s e foram capazes de colocar alguns ovos antes da FUDR entrou em vigor. O FUDR geralmente impedir que as larvas se transformem em adultos completa, mas, ocasionalmente, alguns vão se transformar em adultos e se confundem com os animais experimentais. </li><li> Bactérias vivas / crescentes são observadas. Geralmente, a combinação de Amp e UV-matança vai garantir que nenhuma bactéria contaminar viver a experiência, mas ocasionalmente ocorre. </li><li> Crescimento do fungo é observada na mídia. Se detectada a tempo, o crescimento de fungos geralmente podem ser cortadas usando uma ponteira ou espátula. Uma vez que cresce a ser maior do que alguns milímetros de diâmetro é geralmente mais fácil de transferir os vermes para um novo prato. </li><li> Usando o escopo de dissecação, determinar se cada um worm é vivo ou morto. </li><li> Bata suavemente a placa. O worm é vivo se move em resposta ao toque. </li><li> Se o worm não responde a tocar a placa de zoom, em na região da cabeça. </li><li> Bata suavemente a cabeça do verme com pick transferência de platina. Pontuação o worm como morto, se ele não responder movendo a cabeça. </li><li> Worms mortos pode ser removido da placa. </li></ul></li><li> Registrar a data eo número de vermes que estão vivos e mortos. </li><li> Retorno placas a 20 ° C. </li><li> Repita os passos 3,3 a 3,6 a cada 2 a 3 dias até que todos os vermes já morreram. </li></ol> Parte 4: Resultados Representante. Os dados brutos produzidos por um período de experiência de nematóides de vida é uma lista de datas com números correspondentes de vermes que estão vivos e mortos para cada cepa testada. O número de worms que morrem em cada dia é tipicamente invertido para calcular a proporção de vermes vivos em cada dia (Figura 2A), que é plotado graficamente como uma curva de sobrevivência (Figura 2B, o dia da postura de ovos cronometrado é considerado dia 0 ). O tempo de vida para cada verme individuais no estudo pode ser calculado a partir da contagem de vermes que morrem em cada dia e usado para calcular tempo de vida média e mediana para a comparação entre as linhagens. A contagem do número de vermes vivos em cada dia não é utilizado diretamente na análise de ciclo de vida. Worms mantidos em meios sólidos irá ocasionalmente &#39;fugir&#39;, ou rastrear tanto para cima da parede da placa ou para baixo abaixo da media. O número de vermes vivos em cada dia pode ser usado para determinar quantos worms fugiram durante todo o curso do experimento. Worms que fogem são normalmente removidos da análise. Como referência, o tempo de vida típico mediana para N2, a C. elegans cepa do tipo selvagem, mantidos em UV-matou as bactérias a 20 ° C é de aproximadamente 25 dias, como medida de ovo. Parte 5: Soluções <table border="0"><tbody><tr><td colspan="2"> Nematóide Crescimento Media (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 por 40 minutos e deixe esfriar até 55 ° C, em seguida, acrescentar: </td></tr><tr><td> 100 L </td><td> 1 M MgSO 4 </td></tr><tr><td> 100 L </td><td> Colesterol 5 mg / mL </td></tr><tr><td> 100 L </td><td> 1 M 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 pode ser usado imediatamente para derramar placas ou permissão para solidificar e armazenados a longo prazo na temperatura ambiente </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> Luria Broth (LB), 1L: </td></tr><tr><td> 10 g </td><td> BactoTryptone </td></tr><tr><td> 5 g </td><td> Extrato de levedura </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> água deionizada </td></tr><tr><td colspan="2"> Autoclave e armazenar em 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> MgSO 4 </td></tr><tr><td> 300 mL </td><td> água deionizada </td></tr><tr><td colspan="2"> Autoclave e armazenar em temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> Colesterol 5 mg / mL, 200 mL: </td></tr><tr><td> 1 g </td><td> colesterol </td></tr><tr><td> 200 mL </td><td> Etanol 100% </td></tr><tr><td colspan="2"> Filtro de esterilizar e armazenar à 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> CaCl 2 </td></tr><tr><td> 500 mL </td><td> água deionizada </td></tr><tr><td colspan="2"> Filtro de esterilizar e armazenar à 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> água deionizada </td></tr><tr><td colspan="2"> De calor durante a mistura para permitir KPI para dissolver na solução. Ajustar o pH para 6,0 com NaOH 10 N. </td></tr><tr><td colspan="2"> Adicione água deionizada para 1 L. </td></tr><tr><td colspan="2"> Autoclave e armazenar em 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> água deionizada </td></tr><tr><td colspan="2"> Ajustar o pH para 8,0 com HCl. </td></tr><tr><td colspan="2"> Adicionar água deionizada até 500 mL. </td></tr><tr><td colspan="2"> Filtro de esterilizar e armazenar à temperatura ambiente. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 150 mM Fluorodeoxyuridine (FUDR), 10 mL: </td></tr><tr><td> 0,3693 g </td><td> FUDR </td></tr><tr><td> 10 mL </td><td> água deionizada estéril </td></tr><tr><td colspan="2"> Armazenar a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 100 mg / mL Ampicilina (AMP), 10 mL: </td></tr><tr><td> 1 g </td><td> Ampicilina </td></tr><tr><td> 10 mL </td><td> água deionizada estéril </td></tr><tr><td colspan="2"> Armazenar 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> água deionizada estéril </td></tr><tr><td colspan="2"> Armazenar a -20 ° C. </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1 M isopropílico β-d-Thiogalactopyranoside (IPTG), 10 mL: </td></tr><tr><td> 2,38 g </td><td> IPTG </td></tr><tr><td> 10 mL </td><td> água deionizada estéril </td></tr><tr><td colspan="2"> Armazenar a -20 ° C. </td></tr></tbody></table><img src="/files/ftp_upload/1152/Figure%201.png" alt="figura 1" /> Figura 1. Brilhante imagens campo de C. fases da vida elegans, incluindo ovo, quatro estágios larvais (L1 - L4) e adultos. Todos os painéis mostram hermafroditas, exceto o inferior direito, o que mostra um homem adulto (Imagem de Wood (1988)). <img src="/files/ftp_upload/1152/Figure%202.png" alt="figura 2" /> Figura 2. Os resultados representativos de um C. elegans experimento tempo de vida comparando N2 linhagem selvagem tipo para uma cepa que contém uma mutação no gene daf-2. (A) Uma tabela mostrando os dados coletados, incluindo o número de dias desde worms foram ovos, número de vermes mortos observadas em cada dia, ea porcentagem da amostra original permanecer vivo em cada dia (como calculado a partir da contagem diária de vermes mortos) . (B) Survival curvas correspondentes aos dados de tempo de vida previsto no (A). <imgsrc = &quot;/ files/ftp_upload/1152/Figure 3.jpg&quot; alt = &quot;figura 3&quot; /&gt; Figura 3. Comparação Representante da lipofuscina entre jovens e velhos adulto C. elegans. Imagens de campo claro são mostrados à esquerda e as imagens de fluorescência DAPI canal à direita. Painéis superiores mostram um verme 4 dias de idade e painéis de fundo mostram um worm de 11 dias de idade (medida a partir de ovo).

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	<li>Wood, W. B., Wood, W. B. <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 Nematode Caenorhabditis elegans. , 1-16 (1988).</li><li>Sutphin, G. L., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+by+bacterial+deprivation+increases+life+span+in+wild-derived+nematodes.">Dietary restriction by bacterial deprivation increases life span in wild-derived nematodes.</a> Exp Gerontol. 43, 130-135 (2008).</li><li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans:+a+role+for+heat-shock+factor+and+bacterial+proliferation.">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li><li>Chen, D., Pan, K. Z., Palter, J. E., Kapahi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+determined+by+developmental+arrest+genes+in+Caenorhabditis+elegans.">Longevity determined by developmental arrest genes in Caenorhabditis elegans.</a> Aging Cell. 6, 525-523 (2007).</li><li>Curran, S. P., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+regulation+by+evolutionarily+conserved+genes+essential+for+viability.">Lifespan regulation by evolutionarily conserved genes essential for viability.</a> PLoS Genet. 3, e56-e56 (2007).</li><li>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=Rates+of+behavior+and+aging+specified+by+mitochondrial+function+during+development.">Rates of behavior and aging specified by mitochondrial function during development.</a> Science. 298, 2398-2401 (2002).</li><li>Hamilton, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+for+longevity+genes+in+C.+elegans.">A systematic RNAi screen for longevity genes in C. elegans.</a> Genes Dev. 19, 1544-1555 (2005).</li><li>Hansen, M., Hsu, A. L., Dillin, A., 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=New+genes+tied+to+endocrine,+metabolic,+and+dietary+regulation+of+lifespan+from+a+Caenorhabditis+elegans+genomic+RNAi+screen.">New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen.</a> PLoS Genet. 1, 119-128 (2005).</li><li>Lee, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+identifies+a+critical+role+for+mitochondria+in+C.+elegans+longevity.">A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity.</a> Nat Genet. 33, 40-48 (2003).</li><li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+calorie-independent+life+span+extension+from+dietary+restriction+by+bacterial+deprivation+in+Caenorhabditis+elegans.">Age- and calorie-independent life span extension from dietary restriction by bacterial deprivation in Caenorhabditis elegans.</a> BMC Dev Biol. 8, 49-49 (2008).</li><li>Hodgkin, J., Horvitz, H. R., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+Mutants+of+the+Nematode+CAENORHABDITIS+ELEGANS.">Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS.</a> Genetics. 91, 67-94 (1979).</li><li>Emmons, S. W., Sternberg, P. W., Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. 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 II. , (1997).</li><li>Lipton, J., Kleemann, G., Ghosh, R., Lints, R., Emmons, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mate+searching+in+Caenorhabditis+elegans:+a+genetic+model+for+sex+drive+in+a+simple+invertebrate.">Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate.</a> J Neurosci. 24, 7427-7434 (2004).</li><li>Davis, B. O., Anderson, G. L., Dusenbery, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans.">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</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>Steinkraus, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+suppresses+proteotoxicity+and+enhances+longevity+by+an+hsf-1-dependent+mechanism+in+Caenorhabditis+elegans.">Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans.</a> Aging Cell. 7, 394-404 (2008).</li></ol>

O controle genético da longevidade tem sido estudado extensivamente em C. elegans, em grande parte devido à facilidade e rapidez com que expectativa de vida pode ser determinada. O protocolo discutido neste artigo descreve uma estrutura básica para a obtenção de dados de vida reprodutíveis extensão em C. elegans e também pode ser aplicado às espécies relatednematode. 2 Ao fazer algumas alterações simples desses procedimentos pode ser adaptado para medir a expectativa de vida sob ...

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<td>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0145-17-0 (214530)</td>
<td>&nbsp;</td>
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<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
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<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

O envelhecimento é um processo degenerativo caracterizado por uma deterioração progressiva de componentes celulares e organelas, resultando em mortalidade. O nematóide<em&gt; Caenorhabditis elegans</em&gt; Surgiu como um modelo principal utilizado para estudar a biologia do envelhecimento. Porque praticamente todos os subsistemas biológico sofre declínio funcional com o aumento da idade, expectativa de vida é o objetivo primário de interesse quando se considera taxa total de envelhecimento. Em nematóides, expectativa de vida é normalmente definida como o número de dias de um animal continua a responder a estímulos externos. Nematóides podem ser propagadas tanto em meios líquidos ou em meios sólidos em placas, e as técnicas foram desenvolvidas para medir o tempo de vida em ambas as condições. Aqui apresentamos um protocolo generalizada para medir tempo de vida dos nematóides mantidos em meio de crescimento sólido de nematóides e alimentados com uma dieta de UV-morto bactérias. Estes procedimentos podem ser facilmente adaptado a vida útil do ensaio sob várias condições comuns, incluindo uma dieta composta de bactérias vivas, a restrição alimentar e interferência de RNA.

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

Neste artigo apresentamos um protocolo geral para medir a expectativa de vida dos nematóides mantidos em meios sólidos com alimentos UV-morto bacteriana.

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 ...

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

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Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

29.7K visualizações.  University of Washington. Neste artigo apresentamos um protocolo geral para medir a expectativa de vida dos nematóides mantidos em meios sólidos com alimentos UV-morto bacteriana.

Vídeo: 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.

Quantificar Yeast Life Span cronológica por conseqüência de células envelhecidas

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

Biologia

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.

Medição Span vida replicativa na levedura de brotamento

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.

Medição Caenorhabditis elegans Expectativa de Vida em 96 placas de microtitulação Bem

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 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 ...

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 ...

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.

A cultura<em&gt; Caenorhabditis elegans</em &gt; Em Axenic Liquid Media e Criação de transgênicos Worms por bombardeamento com 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.

Medição de resistência de Estresse Oxidativo<em&gt; Caenorhabditis elegans</em&gt; Em placas de 96 poços de microtitulação

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.

Usando elegans caenorhabditis para tela para interações de acompanhantes específicas de tecido

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.

Imagem livre de rótulos da Dinâmica de Armazenamento Lipídido em Elegans caenorhabditis usando microscopia de dispersão raman estimulada

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

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 ...