Reconhecemos o Centro de Genética Caenorhabditis para C. estirpes elegans. Suporte O financiamento foi fornecido pelo Instituto de Pesquisa Fox Terry. Agradecemos também o apoio concedido à EP da Rolande e Marcel Gosselin Graduate Studentship ea bolsa de formação CIHR / FRSQ em FRN53888 do Programa Integrado de Formação McGill Cancer Research investigação do cancro.

1. Preparação dos Reagentes <ol><li> Preparação da mídia para C. crescimento elegans (neste caso, os animais selvagens e animais flcn-1 (ok975) mutantes). <ol><li> Prepare modificado apenas de Bacto-peptona (BOYM) mistura seca do Youngren contendo 5,5 g de Tris-HCl, 2,4 g de base Tris, 31 g de Bactopetone, 20 g de NaCl e 0,08 g de colesterol. Misture bem com agitação. NOTA: Esta mistura é suficiente para preparar 10 L de meio de MYOB. NOTA: placas normais meio de crescimento (NGM) poderia ser usado em vez de MYOB placas 27. No entanto, o mesmo tipo de placas deve ser utilizada para comparações válidas entre estirpes e entre as repetições independentes. Neste protocolo, nós só usamos placas MYOB. </li><li> Prepare 1 L de meio BOYM por adição de 6 g de BOYM mistura seca, 17 g de ágar e perfazer o volume de 1 litro com H 2 O e autoclave durante 45 min a 122 ° C. Verter em placas e deixar secar à temperatura ambiente durante 2 dias. </li><li> Utilizando uma técnica estéril, inocular 100 ml de culture de meio de caldo LB com E. bactérias coli OP50 e crescer O / N a 37 ° C. </li><li> Placas MYOB semente com E. bactérias coli OP50. Deixe-a crescer à temperatura ambiente durante 48 horas. </li></ol></li><li> Preparação para a regulação negativa do gene usando RNAi <ol><li> Prepare MYOB placas RNAi-alimentação. Proceder como em passos 1.1.1 e 1.1.2. Após autoclavagem, permitir que o meio arrefeça até cerca de 55 ° C e adiciona-se 1 ml de 1 M de isopropilo β-D-1-tiogalactopiranósido (IPTG) e 1 ml de 100 mM de ampicilina. </li><li> Streak E. coli HT115 bactérias transformadas com um plasmídeo que exprime ou EV ou controlo RNAi flcn-1 específico em ampicilina (50 ug / mL) / tetraciclina (15 ug / ml) em placas de agar. Crescer a cultura O / N a 37 ° C. </li><li> Escolha colônias e inocular E. coli HT115 bactérias transformadas com um plasmídeo que exprime ou EV ou controlo flcn-1 RNAi específico e crescer a cultura em LB / ampicilina (50 ug / ml) e meio O / N a 37 &amp; #176; C. </li><li> Placas de sementes com E. coli HT115 EV bactérias ou HT115 flcn-1 bactérias de ARNi. NOTA: Manter placas de 48 horas à temperatura ambiente antes do uso. Se RNAi knockdown por alimentação é eficaz em, utilizar outros métodos de entrega da ARN-DS 28. </li></ol></li><li> Induzir o estresse oxidativo utilizando paraquat (PQ): <ol><li> Preparar tampão M9 por mistura de 3 g de KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl e 0,25 g de MgSO 4 7 · H2O Traga até 1 L com água destilada. Solução em autoclave durante 45 min a 122 ° C e armazenar garrafas à TA. </li><li> No dia do ensaio, preparar a solução de M9 / PQ contendo. Para este protocolo, utilizar PQ 100 mM (0,1028 g de PQ dissolvido em 4 ml de tampão M9 preenche toda uma placas de 96 poços com 40 ul de M9 / PQ por poço). CUIDADO: PQ é um composto altamente tóxico e perigoso. Manusear de acordo com as orientações adequadas. NOTA: Quando a primeira a executar um novo scomboio ou de uma nova condição, recomenda-se a ensaio de sobrevivência em concentrações crescentes de PQ para determinar a melhor concentração de usar, a fim de matar os animais dentro de 7-10 horas. </li></ol></li></ol> 2. Preparação de Age-sincronizado L4 População <ol><li> Transferir 20 hermafroditas adultas grávidas a uma placa de MYOB fresco semeado com E. bactérias coli OP50. Prepare várias placas com base no número de vermes necessários durante o ensaio. </li><li> Permitir-lhes a pôr ovos durante 6 horas e em seguida, usando um worm fio de platina picareta remover as mães. Verificar as placas sob o microscópio para avaliar o número de ovos postos. </li><li> Permitir que os ovos para chocar e crescer a 20 ° C durante 48 h. Neste momento, os animais estão em um L4 / estágio final adulto jovem. Escolha mesmos animais de palco e transferir para os poços contendo a solução PQ. Observação: Uma vez que 48 horas de incubação só se aplica aos mutantes que crescem de forma semelhante aos animais de tipo selvagem, que é importante monitorizar de desenvolvimentotaxa de mutações recentemente testados para garantir que está de acordo com a taxa de desenvolvimento de tipo selvagem, de outra forma, outras linhas de tempo deve ser utilizado. </li><li> Alternativamente, a utilização de técnicas de sincronização. Gerar população sincronizada de vermes larvas L1 utilizando solução de hipoclorito (25 ml de água, 125 ml de hipoclorito de sódio a 5,25%, 50 ml de NaOH 4 M; embrulhar garrafa com folha de alumínio para isolar da luz). No entanto, para este ensaio, não é recomendável, uma vez que o branqueamento intenso pode afectar o comportamento dos animais durante o ensaio. CUIDADO: Manusear solução de hipoclorito de acordo com as diretrizes. NOTA: Ao usar o RNAi para regular para baixo o gene de interesse, sincronizar os hermafroditas, como descrito na seção 2, com excepção da transferência das mães para placas de RNAi semeados com as bactérias específicas de RNAi. Quando o knockdown com o RNAi é fraca, recomenda alimentação para várias gerações. </li></ol> 3. Realização do Estresse Oxidativo Resistência Ensaio &lt;/ P&gt; <ol><li> Pipetar 40 ml da solução de 100 mM de PQ (preparada) a cada poço de uma placa de 96 poços. Utilize pelo menos 12 poços com réplicas de todas as condições (tratamento, mutante, etc.). Usando um verme picareta fio de platina, transferir 5-8 animais larvas L4 para cada bem indicando o horário de início e fim do tempo. </li><li> Ao iniciar uma outra condição, anote o horário de início e fim do tempo. Colocar a placa a 20 ° C no tempo de incubação entre transferindo os vermes e marcando animais mortos uma hora mais tarde a partir da hora de início. </li><li> Utilizando o microscópio de dissecação, a contagem de sobrevivência a cada hora. Agite ligeiramente a placa antes de iniciar a contagem. Indique vermes que não se movem, mesmo após avistar uma luz de alta intensidade, como animais mortos. Além disso, indique o número de animais vivos. NOTA: Olhando forma verme, caudas e movimento da cabeça em alta ampliação, ajudar a determinar de vermes mortos vivos. </li><li> Repita este passo a cada hora até que a maioria dos worms estão mortos. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Determinação da Sobrevivência Percentagem em cada tempo de Ponto <ol><li> Calcular o número total de animais por estado somando o número total de animais transferidas para cada poço. Ignore os animais que estão danificados ou mortos após a transferência. </li><li> Para cada ponto de tempo, calcular o número total de animais mortos por condição (soma de animais mortos nos 12 poços). Para cada ponto de tempo, calcular a percentagem de morte (número de animais mortos, dividido pelo número total de animais e multiplicado por 100) e percentagem de sobrevivência (100 menos a morte por cento). </li><li> Repetir este ensaio pelo menos três vezes independentes. </li></ol>

<ol>
	<li>Schieber, M., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROS+function+in+redox+signaling+and+oxidative+stress.">ROS function in redox signaling and oxidative stress.</a> Curr Biol. 24, R453-R462 (2014).</li><li>Alfadda, A. A., Sallam, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+in+health+and+disease.">Reactive oxygen species in health and disease.</a> J Biomed Biotechnol. 2012, 936486 (2012).</li><li>Finkel, T., Holbrook, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidants,+oxidative+stress+and+the+biology+of+ageing.">Oxidants, oxidative stress and the biology of ageing.</a> Nature. 408, 239-247 (2000).</li><li>Di Carlo, M., Giacomazza, D., Picone, P., Nuzzo, D., San Biagio, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+oxidative+stress+and+mitochondrial+dysfunction+the+key+players+in+the+neurodegenerative+diseases.">Are oxidative stress and mitochondrial dysfunction the key players in the neurodegenerative diseases.</a> Free Radic Res. 46, 1327-1338 (2012).</li><li>Gandhi, S., Abramov, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+oxidative+stress+in+neurodegeneration.">Mechanism of oxidative stress in neurodegeneration.</a> Oxid Med Cell Longev. 2012, 428010 (2012).</li><li>Trushina, E., McMurray, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+mitochondrial+dysfunction+in+neurodegenerative+diseases.">Oxidative stress and mitochondrial dysfunction in neurodegenerative diseases.</a> Neuroscience. 145, 1233-1248 (2007).</li><li>Touyz, R. M., Briones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+vascular+biology:+implications+in+human+hypertension.">Reactive oxygen species and vascular biology: implications in human hypertension.</a> Hypertens Res. 34, 5-14 (2011).</li><li>Gorrini, C., Harris, I. S., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+oxidative+stress+as+an+anticancer+strategy.">Modulation of oxidative stress as an anticancer strategy.</a> Nat Rev Drug Discov. 12, 931-947 (2013).</li><li>Sosa, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+cancer:+an+overview.">Oxidative stress and cancer: an overview.</a> Ageing research reviews. 12, 376-390 (2013).</li><li>Van Raamsdonk, J. M., 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=Reactive+Oxygen+Species+and+Aging+in+Caenorhabditis+elegans:+Causal+or+Casual+Relationship.">Reactive Oxygen Species and Aging in Caenorhabditis elegans: Causal or Casual Relationship.</a> Antioxid Redox Signal. 13, 1911-1953 (2010).</li><li>Baumeister, R., Schaffitzel, E., Hertweck, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+signaling+in+Caenorhabditis+elegans+controls+stress+response+and+longevity.">Endocrine signaling in Caenorhabditis elegans controls stress response and longevity.</a> J Endocrinol. 190, 191-202 (2006).</li><li>Markaki, M., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+in+Caenorhabditis+elegans.">Modeling human diseases in Caenorhabditis elegans.</a> Biotechnol J. 5, 1261-1276 (2010).</li><li>Rodriguez, M., Snoek, L. B., De Bono, M., Kammenga, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Worms+under+stress:+C.+elegans+stress+response+and+its+relevance+to+complex+human+disease+and+aging.">Worms under stress: C. elegans stress response and its relevance to complex human disease and aging.</a> Trends Genet. 29, 367-374 (2013).</li><li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical approach series. , 282 (1999).</li><li>C. elegans Sequencing Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+the+nematode+C.+elegans:+a+platform+for+investigating+biology.">Genome sequence of the nematode C. elegans: a platform for investigating biology.</a> Science. 282, 2012-2018 (1998).</li><li>Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turn+to+the+worm!.">Turn to the worm!.</a> Current opinion in genetics &amp; development. 7, 410-415 (1997).</li><li>Wheelan, S. J., Boguski, M. S., Duret, L., Makalowski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+and+nematode+orthologs--lessons+from+the+analysis+of+1800+human+genes+and+the+proteome+of+Caenorhabditis+elegans.">Human and nematode orthologs--lessons from the analysis of 1800 human genes and the proteome of Caenorhabditis elegans.</a> Gene. 238, 163-170 (1999).</li><li>Culetto, E., Sattelle, 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=A+role+for+Caenorhabditis+elegans+in+understanding+the+function+and+interactions+of+human+disease+genes.">A role for Caenorhabditis elegans in understanding the function and interactions of human disease genes.</a> Hum Mol Genet. 9, 869-877 (2000).</li><li>Fire, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Lamitina, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+approaches+in+C.+elegans.">Functional genomic approaches in C. elegans.</a> Methods Mol Biol. 351, 127-138 (2006).</li><li>Poulin, G., Nandakumar, R., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screens+in+Caenorhabditis+elegans:+impact+on+cancer+research.">Genome-wide RNAi screens in Caenorhabditis elegans: impact on cancer research.</a> Oncogene. 23, 8340-8345 (2004).</li><li>Moreno-Arriola, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans:+A+Useful+Model+for+Studying+Metabolic+Disorders+in+Which+Oxidative+Stress+Is+a+Contributing+Factor.">Caenorhabditis elegans: A Useful Model for Studying Metabolic Disorders in Which Oxidative Stress Is a Contributing Factor.</a> Oxid Med Cell Longev. , 705253 (2014).</li><li>Possik, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folliculin+regulates+ampk-dependent+autophagy+and+metabolic+stress+survival.">Folliculin regulates ampk-dependent autophagy and metabolic stress survival.</a> PLoS Genet. 10, e1004273 (2014).</li><li>Fukushima, T., Tanaka, K., Lim, H., Moriyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+cytotoxicity+of+paraquat.">Mechanism of cytotoxicity of paraquat.</a> Environ Health Prev Med. 7, 89-94 (2002).</li><li>Van Raamsdonk, J. M., 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=Superoxide+dismutase+is+dispensable+for+normal+animal+lifespan.">Superoxide dismutase is dispensable for normal animal lifespan.</a> Proc Natl Acad Sci U S A. 109, 5785-5790 (2012).</li><li>Schulz, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+restriction+extends+Caenorhabditis+elegans+life+span+by+inducing+mitochondrial+respiration+and+increasing+oxidative+stress.">Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress.</a> Cell Metab. 6, 280-293 (2007).</li><li>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=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Timmons, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+methods+for+RNA+interference+in.">Delivery methods for RNA interference in.</a> C. elegans. Methods Mol Biol. 351, 119-125 (2006).</li><li>Wang, B. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+Eyes+Absent+Ortholog+EYA-1+Is+Required+for+Stress+Resistance.">Caenorhabditis elegans Eyes Absent Ortholog EYA-1 Is Required for Stress Resistance.</a> Biochemistry (Mosc). 79, 653-662 (2014).</li><li>Paz-Gomez, D., Villanueva-Chimal, E., Navarro, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DEAD+Box+RNA+helicase+VBH-1+is+a+new+player+in+the+stress+response+in+C.+elegans.">The DEAD Box RNA helicase VBH-1 is a new player in the stress response in C. elegans.</a> PLoS One. 9, 97924 (2014).</li><li>Ward, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defects+in+the+C.+elegans+acyl-CoA+synthase,+acs-3,+and+nuclear+hormone+receptor,+nhr-25,+cause+sensitivity+to+distinct,+but+overlapping+stresses.">Defects in the C. elegans acyl-CoA synthase, acs-3, and nuclear hormone receptor, nhr-25, cause sensitivity to distinct, but overlapping stresses.</a> PLoS One. 9, 92552 (2014).</li><li>Staab, T. A., Evgrafov, O., Knowles, J. A., Sieburth, D. <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+synaptic+nlg-1/neuroligin+abundance+by+the+skn-1/Nrf+stress+response+pathway+protects+against+oxidative+stress.">Regulation of synaptic nlg-1/neuroligin abundance by the skn-1/Nrf stress response pathway protects against oxidative stress.</a> PLoS Genet. 10, e1004100 (2014).</li><li>Greer, E. L., et al. <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.">An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in.</a> C. elegans. Curr Biol. 17, 1646-1656 (2007).</li><li>Allen, E., Walters, I. B., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brivanib,+a+dual+FGF/VEGF+inhibitor,+is+active+both+first+and+second+line+against+mouse+pancreatic+neuroendocrine+tumors+developing+adaptive/evasive+resistance+to+VEGF+inhibition.">Brivanib, a dual FGF/VEGF inhibitor, is active both first and second line against mouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance to VEGF inhibition.</a> Clin Cancer Res. 17, 5299-5310 (2011).</li><li>Apfeld, J., O'Connor, G., McDonagh, T., DiStefano, P. S., Curtis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMP-activated+protein+kinase+AAK-2+links+energy+levels+and+insulin-like+signals+to+lifespan+in+C.+elegans.">The AMP-activated protein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans.</a> Genes Dev. 18, 3004-3009 (2004).</li><li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+AMP-activated+protein+kinase+AAK-2+is+phosphorylated+by+LKB1+and+is+required+for+resistance+to+oxidative+stress+and+for+normal+motility+and+foraging+behavior.">The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior.</a> J Biol Chem. 283, 14988-14993 (2008).</li><li>Honda, Y., Honda, 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+daf-2+gene+network+for+longevity+regulates+oxidative+stress+resistance+and+Mn-superoxide+dismutase+gene+expression+in+Caenorhabditis+elegans.">The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans.</a> FASEB J. 13, 1385-1393 (1999).</li><li>Restif, C., Metaxas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+swimming+motions+of+C.+elegans+worms+with+applications+in+aging+studies.">Tracking the swimming motions of C. elegans worms with applications in aging studies.</a> Med Image Comput Comput Assist Interv. 11, 35-42 (2008).</li><li>Buckingham, S. D., Sattelle, 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=Fast,+automated+measurement+of+nematode+swimming+(thrashing)+without+morphometry.">Fast, automated measurement of nematode swimming (thrashing) without morphometry.</a> BMC Neurosci. 10, 84 (2009).</li></ol>

The authors declare that they have no competing financial interests.

C. elegans é um organismo modelo atractivo para estudar a resistência ao estresse oxidativo geneticamente in vivo, uma vez que podem ser facilmente cultivadas e rapidamente leva a um grande número de descendentes geneticamente idênticos. Vários métodos para medir a resistência de stress oxidativo têm sido anteriormente descrita e que se baseiam na suplementação de placas de cultura com várias fontes de ROS, como a PQ, rotenona, H 2 O 2, e juglona 25,26,29-32....

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

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

Em eucariotas, fosforilação oxidativa ocorrendo na cadeia de transporte de elétrons da mitocôndria é o principal motor da produção de energia na forma de ATP. Espécies reativas de oxigênio (ROS) são um subproduto natural do processo. Apesar do seu importante papel como moléculas de sinalização, excessiva de ROS pode levar a danos no ADN, carbonilação de proteínas, e a oxidação de lípidos. Um desequilíbrio entre a produção de ROS e desintoxicação faz com que o stress oxidativo, o que conduz à depleção da energia, lesão celular, e provoca a morte celular 1,2. O estresse oxidativo contribui para o envelhecimento e para o desenvolvimento de muitas doenças potencialmente fatais, incluindo câncer, diabetes, doenças cardiovasculares e neurodegenerativas 3-9. As células desenvolveram estratégias de defesa enzimáticos e não enzimáticos para manter os níveis de ROS adequadas e proteger os seus constituintes contra dano oxidativo 1,2. Superóxido dismutase (SOD) enzimas agem primeiro a convert superóxido a H 2 O 2, que é posteriormente convertida em água pela catalase ou enzimas peroxidase. Estratégias de defesa não enzimáticas incluem principalmente moléculas que reagem mais rapidamente com ROS, em comparação com macromoléculas celulares, protegendo os componentes celulares essenciais. Apesar do papel protetor da ROS desintoxicação enzimas, algumas moléculas ROS escapar dos mecanismos de defesa antioxidante e levar a danos oxidativos. Detecção, reparação e degradação dos componentes celulares danificados são estratégias essenciais da defesa durante o estresse oxidativo 1,2. Vias de sinalização envolvidas na resistência ao estresse e estresse oxidativo especificamente são altamente evolutivamente conservada 10,11. Ao contrário de experimentos de cultura de células, onde as condições organismal são apenas parcialmente reproduzido, o estudo do estresse oxidativo em organismos modelo 12,13 tem um grande significado. C. elegans é um nematóide de vida livre que pode ser fácil e barata cultured em um gramado bacteriana no meio de ágar. É pequeno no tamanho (cerca de 1 mm de comprimento) e, normalmente, cresce como um hermafrodita auto-fertilizar, o que facilita as manipulações genéticas. Tem um ciclo de vida rápido e uma alta capacidade reprodutiva, produzindo cerca de 300 descendentes por geração, tornando-se uma ferramenta poderosa para executar telas genéticos de larga escala 14. A C. elegans genoma está completamente sequenciado e 40-50% dos genes é previsível que sejam homólogos de genes associados a doenças humanas 15-18. O knockdown de genes de interesse utilizando RNAi é rápida e fácil em C. elegans. Gene para baixo regulamento poderia ser alcançado através da alimentação de animais a E. bactérias coli que abrigam um plasmídeo que expressa o ARN de cadeia dupla que tem como alvo o mRNA de interesse 19. Portanto, a determinação da função do gene usando telas de RNAi em grande escala tem um grande impacto na compreensão das doenças humanas, incluindo câncer 20,21. Estudos de Oresistência ao estresse xidative em C. elegans levaram à identificação de mecanismos conservadas de resistência ao stress oxidativo 13,22. Algumas vias são identificadas vias comuns que modulam a longevidade e resistência a outros tipos de stress, bem como a hipóxia, calor, e stress osmótico. Estas vias incluem a sinalização de insulina, a sinalização TOR, e autofagia. Outras vias importantes envolvem desintoxicação de ROS como enzimas superóxido dismutase e enzimas catalase, ou na reparação de danos, tais como proteínas de choque térmico e acompanhar 11,13,22. Este protocolo descreve como para determinar a resistência ao stress oxidativo de C. elegans em líquido. Nós usamos flcn-1 (ok975) e animais de tipo selvagem para demonstrar o protocolo já que mostramos anteriormente um aumento da resistência ao estresse oxidativo após a perda de flcn-1 (ok975) em C. elegans 23. Também mostraram que este aumento da resistência dependeem AMPK e autofagia, um eixo de sinalização que melhora bioenergética celular e promove a resistência ao estresse 23. PQ é um estressor oxidativo que interfere com a cadeia de transporte de elétrons para produzir espécies reativas de oxigênio 24. O mesmo ensaio pode ser adaptado e outras fontes de ROS ou compostos que geram ROS poderia ser utilizado tal como H 2 O 2 e rotenona. Ensaios semelhantes foram desenvolvidas em placas onde as baixas concentrações de PQ são utilizados 25,26. A vantagem deste teste é que ele é muito rápido, e os resultados podem ser obtidos em apenas um dia. Além disso, o volume total de líquido utilizado para realizar o ensaio de resistência ao estresse oxidativo em placas de 96 poços é baixo quando comparado com o volume utilizado para preparar-PQ contendo placas. Portanto, a quantidade de PQ usado é no ensaio de líquido é baixa, o que torna o ensaio pouco dispendioso e limita a produção de resíduos tóxicos. No entanto, limitações deste ensaio em comparação com ensaios em placa incluem o lack de alimento no líquido de ensaio e a menor concentração de oxigénio no líquido, em comparação com o ar. Estes são factores importantes que, em alguns casos, podem influenciar os resultados. Portanto, confirmando a reprodutibilidade utilizando outros métodos de resistência ao estresse oxidativo é recomendado para suportar os resultados obtidos neste ensaio.

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

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

O stress oxidativo, que é o resultado de um desequilíbrio entre a produção e a desintoxicação de espécies reactivas de oxigénio, é um dos principais contribuintes para doenças humanas crónicas, incluindo doenças cardiovasculares e neurodegenerativas, diabetes, envelhecimento e cancro. Portanto, é importante para estudar o stress oxidativo, não só em sistemas de células, mas também utilizando organismos inteiros. C. elegans é um organismo modelo atraente para estudar a genética de vias de transdução de sinal de estresse oxidativo, que são altamente conservadas evolutivamente. Aqui, nós fornecemos um protocolo para medir a resistência ao estresse oxidativo em C. elegans em líquido. Reagentes Resumidamente, ROS-indutores, tais como paraquato (PQ) e H 2 O 2 são dissolvidos em tampão M9, e soluções são aliquotadas em cavidades de uma placa de microtitulação de 96 poços. L4 sincronizado / adulto jovem C. elegans animais são transferidos para os poços (5-8 animais / poço) e é medida a sobrevivênciaa cada hora até que a maioria dos worms estão mortos. Ao realizar um ensaio de resistência ao estresse oxidativo utilizando uma baixa concentração de estressores em placas, o envelhecimento pode influenciar o comportamento dos animais sobre o estresse oxidativo, o que poderia levar a uma interpretação incorrecta dos dados. No entanto, no ensaio aqui descrito, este problema é improvável de ocorrer uma vez que apenas L4 / animais adultos, jovens estão sendo usados. Além disso, este protocolo é barato e resultados são obtidos em um dia, o que torna esta técnica atraente para telas genéticos. No geral, isso irá ajudar a compreender vias de transdução de sinal de estresse oxidativo, que poderia ser traduzido em melhor caracterização de doenças humanas associadas a estresse oxidativo.

Comparando-tipo selvagem C. elegans para flcn-1 (ok975) animais mutantes Aqui utilizou-se 100 mM de PQ para determinar a resistência de tipo selvagem C. elegans animais em comparação com flcn-1 (ok975), que foi mostrado para resistir ao estresse oxidativo, calor e anoxia 23. Após 4 h de tratamento, 48,3% da do tipo selvagem sobreviveram, em comparação com 77,8% de sobrevivência em-um (flcn ok975) animais. Como esperado, flcn-...

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

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

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

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 ...

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

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

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

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

Measuring Caenorhabditis elegans Life Span on Solid Media

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

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 ...

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 ...

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 ...

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

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

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

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

Ice Cap: Um Método para Crescer Arabidopsis Plantas e tomate em placas de 96 poços para High-Throughput Genotipagem

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: 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, ...

Analysis of Oxidative Stress in Zebrafish Embryos

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

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

Análise do estresse oxidativo em embriões Zebrafish

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

cultivo de<em&gt; Caenorhabditis elegans</em&gt; Em três dimensões em Laboratório

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

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

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 ...