Gostaríamos de agradecer a S. Taneja-Bageshwar, K. Beifuss, S. Kedroske, e HC. Hsiao para discussões úteis. Este trabalho foi financiado por fundos start-up do Departamento TAMHSC de Medicina Molecular e Celular. O escopo composto e disco giratório foram comprados com fundos fornecidos pelo departamento eo Escritório TAMHSC do Dean. Algumas cepas foram fornecidos pelo Centro de Genética Caenorhabditis, que é financiado pelo Centro Nacional de Pesquisa de Recursos. pRF4 (rol-6 (su1006)) foi um presente de Fogo A..

1. Preparação de DII mancha <ol><li> Prepare uma solução estoque de 20 mg / mL DII (Biotium, Inc., Hayward, CA) em DMF. DII é sensível à luz, de modo a proteger da luz DII por envolvimento em folha. </li><li> Criar uma diluição de trabalho de DII, adicionando 0,6 mL de ações DII para 399,4 M9 mL para cada população. Isso deve dar uma diluição final de trabalho de 30 mcg / mL em DII M9. Isto pode ser ampliada para a coloração de múltiplas populações simultaneamente. DII escudo da luz envolvendo o tubo (s) em folha. </li></ol> 2. Preparação de nematóides <ol><li> Use uma placa de 60 milímetros contendo uma população de nematóides não contaminada. Lavar os animais de placa usando uma solução de 0,5% Triton X-100 em tampão M9 rodando suavemente líquido em um movimento circular em toda a superfície da placa para soltar todos os animais de larva e adulto. Transferência de lavar em um tubo estéril 1,5 mL. </li><li> Imediatamente girar os animais a 2000 rpm por 30 segundos. Remova e descarte o máximo supernatant possível sem perturbar a massa de animais no fundo do tubo. </li><li> Para reduzir residual Triton X-100, lavar os animais usando M9 buffer, spin, e remover o sobrenadante. Repita este passo uma vez. </li><li> Adicionar 400 mL de solução de trabalho DII em M9 para o tubo e agitar brevemente em vórtice para ressuspender animais na solução. </li><li> Agite tubo a 20 ° C na horizontal durante 3 horas a 350 rpm em um ambiente de luz protegida. Se desejado, os animais podem ser incubadas até 16 horas para a coloração. </li><li> Para reduzir a quantidade de tintura não acoplado, girar os animais a 2000 rpm por 20 seg. Remova e descarte o máximo possível sobrenadante sem perturbar a massa de animais. </li><li> Animais ressuspender em 400 mL de buffer M9 e derramar líquido sobre uma parte livre de bactérias de uma placa de agar NGM semeados com OP50 E. coli. Permitir que os animais para recuperar pelo menos 30 minutos. Durante o tempo de recuperação dos animais deve arrastar para longe o líquido coloração DII e para a comida. Este passoreduz a fluorescência de fundo de DII livre. </li></ol> 3. Montagem e observação de espécimes <ol><li> Melt agar 4% em água usando um autoclave ou forno de microondas. </li><li> Criar espaçadores reutilizáveis, que podem ser usados ​​para garantir a espessura uniforme da almofada agar, por camadas dois pedaços de fita laboratório em uma lâmina de vidro. Faça dois slides espaçador total. </li><li> Organizar uma lâmina de vidro limpa entre dois slides espaçador. Pipetar cerca de 150 mL (quatro gotas) de 4% agar derretido para o centro da lâmina de vidro limpa. Rapidamente cobrir o ágar fundido usando um slide adicional para formar um bloco de agar. Remova cuidadosamente a cobertura deslizante, mantendo o bloco centrado na parte superior do slide de montagem. </li><li> Pipetar cerca de 5 mL de nematóide anestésico (100 M - 1 levamisole mM, por exemplo) sobre a almofada. </li><li> Monte 8-12 animais no anestésico e cobrir com uma lamela de microscópio. </li><li> Observe os animais utilizando um microscópio composto ou confocal equipados com pelo menos uma objetividade 40x<html> ve e um DSRed / TRITC (ou outro compatível) do filtro. O máximo de excitação de fluorescência de DII é 549 nm e sua emissão máxima é 565 nm para corante ligado (Biotium, Inc., Hayward, CA). </li></ol> 4. Resultados representante Manchas DII cutícula do C. do tipo selvagem e mutante elegans. A superfície cuticular contém anéis separados por sulcos circunferenciais e, em alguns estágios, sulcos longitudinais chamado asa. Cada estágio de desenvolvimento tem estruturas cuticulares com composições distintas 2. Sulcos ou sulcos de ambos asa e anéis fluorescente mancha, dependendo da composição da superfície, ao longo de fases de larva e adulto e permanecem visíveis até um dia após a recuperação usando esse método. Manchas fundo fluorescente são por vezes observados (Figuras 2F, G), mas não rotineiramente (Figura 1, Figura 2A-E, H, I). Todas as imagens foram tiradas com disco girando confocal ou, quando indicado, campo amplo de microscopia composto (wf). ent &quot;&gt; <img alt="Figura 1" src="/files/ftp_upload/3362/3362fig1.jpg" /> Figura 1. Manchas DII cutícula e neurônios expostos ambientalmente. L2 animais palco. Ampliação de 630x. Partes da imagem em mosaico foram capturados usando-iVision Mac software (BioVision Technologies, Exton, PA). As imagens foram unidas com Adobe Photoshop CS3 (Adobe Systems, Inc., San Jose, CA). Barra de escala = 10 mM. DII também fluorescente manchas amphid e phasmid neurônios sensoriais na cabeça e cauda, ​​respectivamente (setas marcar alguns). <img alt="Figura 2" src="/files/ftp_upload/3362/3362fig2.jpg" /> Figura 2. DII fluorescente manchas C. elegans cutícula em todas as fases de desenvolvimento pós-embrionário. Ampliação de 630x. Barra de escala = 10 mM. De coloração de tipo selvagem animais em A L1); B) L2; C) Dauer; D) L3; E) L4, e F) Ciclo de adultos. As cristas asa e anular são manchadas fluorescente nos animais L1 e Dauer (A, C). Manchas DII cristas anelares em animais L2(B). Sulcos anulares mancha em L3 e L4 animais (D, E). Os sulcos da asa e anéis estão manchadas em animais adultos (FH). Asa são compostas de dois, cinco, ou três cumes (em L1, Dauer, ou animais adultos, respectivamente) que correm o comprimento do animal (setas) 16. Anéis criar sulcos circunferenciais ao redor do animal (setas F e J). A cutícula de adultos animais mutantes mostrar moderada defeitos organização cuticular (GH). G) Cumes da asa são descontínuos (wf). H) cumes alae supranumerários são fundidos e ramificada ou bifurcada (wf). Mutantes do gene colágeno exibem asa e defeitos organização anular (IJ). I) Em animais transgênicos com superexpressão pRF4 (rol-6 (su1006)), cumes de mentira asa em um ângulo para o comprimento do animal. J) anéis em animais transgênicos com superexpressão pRF4 (rol-6 (su1006)) apresentam um padrão irregular. <img alt="Figura 3" src="/files/ftp_upload/3362/3362fig3.jpg" /> Figura 3. Externestruturas morfológicas al são iluminados por coloração DII. ampliação 630x. Barra de escala = 10 mM. DII também destaca outras características exteriores, incluindo A) adulto hermafrodita vulva, B) adulto raios da cauda dos machos e fã, e C) hermafrodita cauda pico (bifurcada neste contexto mutante). <img alt="Figura 4" src="/files/ftp_upload/3362/3362fig4.jpg" /> Cutícula Figura 4. Demora mais tempo a mancha com DII do que neurônios expostos ambientalmente. Ampliação de 630x. Barra de escala = 10 mM. Após duas horas de coloração, amphid (não mostrado) e phasmid neurônios sensoriais (setas) são suficientemente manchada. Em contraste, a cutícula dos animais mais jovens é apenas parcialmente corados em patches (seta). <table border="1"><tbody><tr><td> Lavar </td><td> Solução mancha (+ DII) </td><td> Tempo de incubação </td><td> Cutícula manchada </td></tr><tr><td> M9 + Tr 0,5%iton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 2 hrs </td><td> não </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 + 0,5% Triton X-100 </td><td> 3 hrs </td><td> não </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 2 hrs </td><td> parcial </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> M9 </td><td> 3 hrs </td><td> sim </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 2 hrs </td><td> parcial </td></tr><tr><td> M9 + 0,5% Triton X-100 </td><td> H 2 0 </td><td> 3 hrs </td><td> sim </td></tr></tbody></table> Tabela 1. Coloração cuticulares em diferentes condições. Vários soluções de incubação e os tempos foram testados para otimizar a coloração cuticular em animais. H 2 0, água destilada estéril. Coloração parcial indica coloração irregular da cutícula larval (Figura 4), embora as manchas cutícula adulto consistently.

<ol>
	<li>Cox, G. N., Kusch, M., Edgar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+of+Caenorhabditis+elegans:+its+isolation+and+partial+characterization.">Cuticle of Caenorhabditis elegans: its isolation and partial characterization.</a> The Journal of Cell Biology. 90, 7-17 (1981).</li><li>Cox, G. N., Staprans, S., Edgar, R. 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+cuticle+of+Caenorhabditis+elegans.+II.+Stage-specific+changes+in+ultrastructure+and+protein+composition+during+postembryonic+development.">The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development.</a> Dev. Biol. 86, 456-470 (1981).</li><li>Hall, D., Altun, Z. <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 Atlas. , (2008).</li><li>Page, A. P., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuticle.">The cuticle.</a> The C. elegans Research Community. , (2007).</li><li>Kramer, J. M., Johnson, J. J., Edgar, R. S., Basch, C., Roberts, 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+sqt-1+gene+of+C.+elegans+encodes+a+collagen+critical+for+organismal+morphogenesis.">The sqt-1 gene of C. elegans encodes a collagen critical for organismal morphogenesis.</a> Cell. 55, 555-565 (1988).</li><li>Mende, N. v. o. n., Bird, D. M., Albert, P. S., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dpy-13:+a+nematode+collagen+gene+that+affects+body+shape.">dpy-13: a nematode collagen gene that affects body shape.</a> Cell. 55, 567-576 (1988).</li><li>Blaxter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+surface+proteins+of+wild+type+and+mutant+Caenorhabditis+elegans.">Cuticle surface proteins of wild type and mutant Caenorhabditis elegans.</a> The Journal of Biological Chemistry. 268, 6600-6609 (1993).</li><li>Costa, M., Draper, B. W., 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=The+role+of+actin+filaments+in+patterning+the+Caenorhabditis+elegans+cuticle.">The role of actin filaments in patterning the Caenorhabditis elegans cuticle.</a> Dev. Biol. 184, 373-384 (1997).</li><li>Sapio, M. R., Hilliard, M. A., Cermola, M., Favre, R., Bazzicalupo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zona+Pellucida+domain+containing+proteins,+CUT-1,+CUT-3+and+CUT-5,+play+essential+roles+in+the+development+of+the+larval+alae+in+Caenorhabditis+elegans.">The Zona Pellucida domain containing proteins, CUT-1, CUT-3 and CUT-5, play essential roles in the development of the larval alae in Caenorhabditis elegans.</a> Dev. Biol.. 282, 231-245 (2005).</li><li>Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cuticle+collagen+genes.+Expression+in+Caenorhabditis+elegans.">Cuticle collagen genes. Expression in Caenorhabditis elegans.</a> Trends Genet. 16, 21-27 (2000).</li><li>Kramer, J. M., French, R. P., Park, E. C., Johnson, J. J. <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+rol-6+gene,+which+interacts+with+the+sqt-1+collagen+gene+to+determine+organismal+morphology,+encodes+a+collagen.">The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.</a> Mol. Cell Biol. 10, 2081-2089 (1990).</li><li>Thein, M. C. <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+exoskeleton+collagen+COL-19:+an+adult-specific+marker+for+collagen+modification+and+assembly,+and+the+analysis+of+organismal+morphology.">Caenorhabditis elegans exoskeleton collagen COL-19: an adult-specific marker for collagen modification and assembly, and the analysis of organismal morphology.</a> Dev. Dyn. 226, 523-539 (2003).</li><li>McMahon, L., Muriel, J. M., Roberts, B., Quinn, M., Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+sets+of+interacting+collagens+form+functionally+distinct+substructures+within+a+Caenorhabditis+elegans+extracellular+matrix.">Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix.</a> Molecular Biology of the Cell. 14, 1366-1378 (2003).</li><li>Link, C. D., Ehrenfels, C. W., 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=Mutant+expression+of+male+copulatory+bursa+surface+markers+in+Caenorhabditis+elegans.">Mutant expression of male copulatory bursa surface markers in Caenorhabditis elegans.</a> Development. 103, 485-495 (1988).</li><li>Tong, Y. G., Burglin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditions+for+dye-filling+of+sensory+neurons+in+Caenorhabditis+elegans.">Conditions for dye-filling of sensory neurons in Caenorhabditis elegans.</a> J. Neurosci. Methods. 188, 58-61 (2010).</li><li>Singh, R. N., Sulston, 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=Some+observations+on+moulting+in+Caenorhabditis+elegans.">Some observations on moulting in Caenorhabditis elegans.</a> Nematologica. 24, 63-71 (1978).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148, 187-200 (1998).</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></ol>

Não temos nada a revelar.

O método de coloração DII aqui apresentado permite uma forma relativamente rápida e conveniente de visualizar a cutícula em C. elegans. Pela redefinição e otimização de um método comumente usado para a imagem ambientalmente expostos neurônios sensoriais 15,17, DII pode ser usado para fluorescente mancha tanto asa e estruturas anulares (Figuras 1 e 2), bem como a vulva, a cauda do sexo masculino e hermafrodita cauda pico (Figura 3). Descobrimos que a solução de incubação ea influência ...

<table border="1"><tbody><tr><td> Reagentes </td><td> Sinônimos </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> DII </td><td> 1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato </td><td> Biotium, Inc. </td><td> 60010 </td><td> Diluição Stock: 20 mg / mL em DMF diluição de trabalho: 30 mg / mL </td></tr><tr><td> DMF </td><td> Dimetilformamida </td><td> Sigma-Aldrich, Inc. </td><td> D4551 </td><td></td></tr><tr><td> Tritão X-100 </td><td> Octylphenoxypolyethoxyethanol </td><td> VWR International, LLC. </td><td> EM-9410 </td><td></td></tr><tr><td> M9 </td><td> KH 2 PO 4 22mm, 42mm Na 2 HPO 4, 86mm NaCl, 1mM MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ Td&gt; <td> Meio de crescimento de nematóides </td><td> IPM Scientific, Inc. </td><td> 11006-501 </td><td> Podem ser preparados seguindo o protocolo NGM ágar 18 </td></tr><tr><td> Ágar-ágar </td><td></td><td> EMD Chemicals, Inc. </td><td> 1,01614 </td><td> 4% em água </td></tr><tr><td> Levamisol </td><td> Cloridrato de levamisol </td><td> Sigma-Aldrich, Inc. </td><td> 31742 </td><td> 100 M - 1 levamisole mM, conforme exigido </td></tr><tr><td> Lâminas de microscópio </td><td></td><td> VWR International, LLC. </td><td> 16005-106 </td><td></td></tr><tr><td> Cobrir copos microscópio </td><td></td><td> VWR International, LLC. </td><td> 16004-302 </td><td></td></tr><tr><td> Âmbito composto </td><td></td><td> Carl Zeiss, Inc. </td><td> A1m </td><td> Use objetivos para corresponder às necessidades do experimento </td></tr><tr><td> TRITC ou outro filtro compatível </td><td> &amp;nbsp; </td><td> Chroma Technology Corp </td><td> 49005 </td><td> ET - DSRed (TRITC/Cy3) sputtered conjunto de filtros </td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

A cutícula do C. elegans é uma estrutura altamente resistente que envolve o exterior do 1-4 animal. A cutícula não só protege o animal do meio ambiente, mas também determina a forma do corpo e desempenha um papel na motilidade 4-6. Várias camadas secretadas por células epidérmicas compreendem a cutícula, incluindo uma camada lipídica externa 7. Sulcos circunferenciais na cutícula chamados anéis padrão o comprimento do animal e estão presentes durante todas as fases de desenvolvimento 8. Asa são sulcos longitudinais, que estão presentes durante fases específicas de desenvolvimento, incluindo L1, Dauer e estágios adultos 2,9. Mutações em genes que afetam a organização do colágeno cuticular pode alterar a estrutura cuticular e morfologia do corpo de animais 5,6,10,11. Enquanto imagem cuticular usando microscopia óptica composto com DIC é possível, os métodos atuais que destacam estruturas cuticulares incluem fluorescentecento expressão do transgene 12, coloração de anticorpos 13, e microscopia eletrônica de 1. Rotulados aglutinina de gérmen de trigo (WGA) também tem sido usado para visualizar glicoproteínas cuticular, mas é limitada na resolução de estruturas mais finas cuticular 14. Coloração da superfície cuticular usando corante fluorescente tem sido observado, mas nunca caracterizados em detalhe 15. Nós apresentamos um método para visualizar cutícula ao vivo C. elegans utilizando o corante vermelho fluorescente lipofílico DII (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato), que é comumente usado em C. elegans para visualizar os neurônios expostos ambientalmente. Este protocolo otimizado para a coloração de DII é um método simples e robusta para a visualização de alta resolução fluorescentes de anéis, asa, vulva, da cauda dos machos, e cauda hermafrodita pico de C. elegans.

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

Nós apresentamos um método para visualizar cutícula ao vivo<em&gt; C. elegans</em&gt; Usando o corante vermelho fluorescente lipofílico DII (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato), que é comumente usado em<em&gt; C. elegans</em&gt; Para visualizar os neurônios expostos ambientalmente. Com este protocolo otimizado, asa e anular estruturas cuticulares estão manchadas pelo DII e observados através de microscopia composto.

Visualização de Caenorhabditis elegans Estruturas cutícula Usando o Dye lipofílicos Vital, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

visualization-caenorhabditis-elegans-cuticular-structures-using

Research

JoVE Journal

Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K visualizações.  Texas A&M University System Health Science Center, College of Medicine. Nós apresentamos um método para visualizar cutícula ao vivo C. elegans Usando o corante vermelho fluorescente lipofílico DII (1,1 '-dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine perclorato), que é comumente usado em C. elegans Para visualizar os neurônios expostos ambientalmente. Com este protocolo otimizado, asa e anular estruturas cuticulares estão manchadas pelo DII e observados através de microscopia composto.

Vídeo: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

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 ...

Biologia

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

A perfusão DiI como método de visualização vascular em<em&gt; Ambystoma mexicanum</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

Abordagens quantitativas para o estudo de estruturas celulares e morfologia organelle em Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

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, ...

Hand Dissection of Caenorhabditis elegans Intestines

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, aging, immunity, and environmental response. Critical interactions between the C. elegans host and its environment converge within the intestine, where gut microbiota concentrate. Therefore, the ability to isolate intestine tissue away from the rest of the worm is necessary to assess intestine-specific processes. This protocol describes a method for hand dissecting adult C. elegans intestines. The procedure can be performed in fluorescently labeled strains for ease or training purposes. Once the technique is perfected, intestines can be collected from unlabeled worms of any genotype. This microdissection approach allows for the simultaneous capture of host intestinal tissue and gut microbiota, a benefit to many microbiome studies. As such, downstream applications for the intestinal preparations generated by this protocol can include but are not limited to RNA isolation from intestinal cells and DNA isolation from captured microbiota. Overall, hand dissection of C. elegans intestines affords a simple and robust method to investigate critical aspects of intestine biology.

Hand Dissection of Caenorhabditis elegans Intestines

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Dissecção da Mão de Caenorhabditis elegans Intestinos

Comprised of only 20 cells, the Caenorhabditis elegans intestine&#160;is the nexus of many life-supporting functions, including digestion, metabolism, ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

Isolamento e Caracterização da Microbiota Natural do Nematoide Modelo Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

Microscopia de super-resolução do complexo sinaptonemal dentro da linha germinal de Caenorhabditis elegans

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...