Este trabalho foi financiado pelos Institutos Nacionais de HealthGrants DK85035 e DK074797 (IH).

1. Cepas Worm <ol><li> Obter C. elegans do tipo selvagem estirpes Bristol N2 a partir do Centro de Genética Caenorhabditis (CGC) (http://www.cbs.umn.edu/cgc) e mantê-los em placas NGM semeados com E. OP50 coli 7. Nota: verme transgênico cepas IQ6011 (Phrg-1 :: GFP) utilizados foram gerados como descrito anteriormente 11. IQ6011 pode ser solicitado ao autor correspondente. </li></ol> 2. Preparação de modificação C. elegans Habitação e Reprodução Médio (mCeHR) Prepare mCeHR meios líquidos, como descrito abaixo de 12. Esta mídia líquido axênica permite que os vermes a crescer sem quaisquer fontes de alimentos adicionais. Realizar todas as manipulações dos meios líquidos axênicas e worms axênicos usando condições rigorosamente estéreis, como uma capela de fluxo laminar. <ol><li> Obter leite livre de gordura ultra-pasteurizado de uma mercearia. Use o pró refrigeradoduto e não o produto a temperatura ambiente. Antes de ser utilizado na mCeHR meio, a sequência de leite em Brain Heart Infusion (BHI) em placas de agar e incubar durante 3 dias a 30 ° C e 37 ° C para verificar a esterilidade. Colocar o leite para tubos de 50 ml estéreis e armazenar a -80 ° C </li><li> Prepare a cada um dos seguintes componentes e combinar na ordem e quantidades apresentadas para preparar 1 litro de mCeHR (Tabela 1A): 10 ml de 2 mM de citrato de colina diácido, 10 ml de vitaminas e mistura de factor de crescimento (ver receita na Tabela 1B), 10 ml de 2,4 mM de mio-inositol, 10 ml de 2 mM de cloreto de hemina, 250 ml de água deionizada. Aplicar sucção e filtra-se através de uma unidade de filtro de 0,22 um. </li><li> Adicionar 20 ml da mistura de ácido nucleico (receita na Tabela 1C), 100 ml de mistura de minerais (receita na Tabela 1E), 20 ml de 170 mg / ml de hidrolisado de lactalbumina, 20 ml de aminoácidos essenciais, 10 ml de aminoácidos não essenciais Os ácidos, de 20 ml de 450 mM de KH 2 PO 4,50 ml de 1,45 M de D-glucose, 10 ml de HEPES 1 M, sal de sódio, 250 ml de água desionizada. </li><li> Aplicar sucção para filtrar esterilizar os componentes. Retire o filtro e adicionar 1 ml de 5 mg / ml de colesterol. Certifique-se que o pH do meio é de cerca de 6-6,5. Nota: Esta solução pode ser armazenada a -80 ° C durante até um ano. </li><li> Adicionar 20% (v / v), ultra-pasteurizado de leite desnatado orgânico para o meio mCeHR antes da utilização. Use técnica escrupulosamente estéril (por exemplo, numa câmara de fluxo laminar), quando a abertura e alíquotas do leite. Armazene a mídia a 4 ° C. </li></ol> 3. Prepare C. elegans para a Cultura em Axenic mCeHR Líquido Médio <ol><li> Crescer vermes em dez 60 milímetros placas NGM até há muitos vermes grávidas e uma quantidade mínima de OP50 E. coli nas placas. </li><li> Remover as minhocas de cada placa por enxaguamento com 5 ml de tampão M9 (receita na Tabela 1F), e combinar num tubo de 50 ml. </li><li> Permita que os vermes para settle, deixando o tubo em repouso durante 5 a 10 min, em seguida, remover cuidadosamente o sobrenadante contendo a E. coli. </li><li> Repita os passos 3.2 e 3.3 2x para remover tanto as bactérias residuais quanto possível, antes de continuar o procedimento. </li><li> Ressuspender as minhocas em 0,1 N de NaCl num volume que é um múltiplo de três, por exemplo, 1,5 ml, 3 ml ou 6 ml, em que os vermes individuais pode ser visto no tubo. </li><li> Branquear os vermes por adição de NaOH 5 N e de hipoclorito de sódio a 5% solução (lixívia) numa proporção de 01:02:06 para a suspensão verme. Por exemplo, 1 ml de NaOH 5 N: 2 ml de hipoclorito de sódio a 5%: 6 ml de suspensão de vírus. Misture o NaOH e branqueador antes da adição à suspensão de verme. </li><li> Vortex se vigorosamente a solução até as minhocas grávidas são dissolvidos e apenas os ovos são visíveis dentro da suspensão (cerca de 5 a 10 min). Monitorar o processo usando um microscópio de contraste de fase com uma objetiva de 10X. </li><li> Após os vermes se ter dissolvido completamente, a pelota imediatamenteovos de 800 g durante 45 segundos a 4 ° C. Remover o sobrenadante e lavar o sedimento duas vezes com 10 ml de água estéril, por centrifugação a pelota em 800 xg durante 45 seg a 4 ° C. </li><li> Após o branqueamento, transferir o sedimento de ovo a um frasco de 25 de tecido cm 2 de cultura contendo 10 ml de meio mCeHR suplementadas com 100 ug / ml de tetraciclina. Realizar este procedimento usando técnicas estéreis. Se for o caso, adicionar antibióticos adicionais, incluindo 250 ug / ml de ácido nalidíxico, para prevenir o crescimento bacteriano nas culturas líquidas axênicos. </li><li> Incubar as culturas liquidas a 20 ° C num incubador com agitação a cerca de 70 rpm. </li><li> Verificar os vermes diária observar a fase de desenvolvimento e a taxa de crescimento. Nota: Worms inicialmente crescem lentamente e exigem 7 a 10 dias para se tornar grávido. No entanto, como os vermes aclimatar ao meio líquido o tempo de geração irá diminuir a aproximadamente 4 dias para o tipo selvagem (N2) vermes. </li><li> Depois que os vermes têm desenvolvido para a fase grávida em meio líquido, a transferência da cultura para um tubo sem-fim cónico e sedimentar as minhocas a 800 xg por 5 min a 4 ° C utilizando um rotor de balde oscilante. </li><li> Remover o sobrenadante e ressuspender cuidadosamente o sedimento de vírus num volume igual de 0,1 M de NaCl. Por exemplo, utilizar 10 ml de 0,1 M de NaCl para vermes cultivados em 10 ml de mCeHR. Permita que os vermes que se contentar com 5 min no gelo. </li><li> Realizar o procedimento de clareamento, como descrito acima em passos 3,5-3,8, e permitir que os vermes a crescer para uma segunda geração nos meios líquidos axênicas. Se necessário, adicionar o antibiótico de novo para inibir o crescimento bacteriano. </li><li> Novamente permitir que os vermes se tornar prenhe então granulada e ressuspender os vermes em 0,1 M de NaCl, tal como descrito nos passos 3.12 e 3.13, então lixívia novamente como descrito nos passos de 3,5-3,8, para produzir uma população sincronizada. Crescer os vermes larvais L1 em ​​meios líquidos sem antibióticos a partir de agora. </li></ol> 4. Sincronizando Worms de líquidoCultura <ol><li> Pellet e ressuspender os vermes em NaCl 0,1 M, como descrito acima nos passos 3.12 e 3.13. Lixívia como descrito acima nos passos 3,5-3,8. </li><li> Ressuspender o sedimento de ovo em 10 ml de tampão M9 e permitir que as larvas eclodem O / N a 20 ° C num agitador de plataforma rotativa. Nota: Os ovos eclodem em larvas L1, mas não vai desenvolver ainda mais, sincronizando os vermes na fase L1. </li><li> Para manter e vermes subcultura, pelota larvas L1 a 800 xg durante 5 min, em seguida, voltar a suspender as larvas em 10 ml de mCeHR e transferência para um frasco de 25 cm 2. Permitir que os vermes a crescer a 20 ° C numa plataforma rotativa. Manter uma densidade máxima de 3.000 vermes / ml / cm 2 para garantir nutrição adequada para os vermes. Nota: Nesta fase, os antibióticos não são necessárias para manter as condições axénicas, quando os procedimentos são efectuados utilizando técnicas esterilizadas numa hote de fluxo laminar. Worms deve ser verificado diariamente para acompanhar o crescimento. </li></ol> 5. Grátiszing e descongelamento Worms para culturas Axenic Médio <ol><li> Congelar culturas vermes assíncronos axênicos ou larvas L1 sincronizado em nitrogênio líquido. Vermes pelete a 800 x g durante 5 min e em seguida ressuspender em tampão S (6,45 mM KHPO 4, 43,55 mM KHPO 4, NaCl 146,25 mM) usando 0,5 ml para cada frasco. Adicionar um volume igual de tampão de S com 30% v / v de glicerol a cada frasco e transferir os vermes para -80 ° C, antes de armazenamento a longo prazo em N2 líquido. Congelar a cerca de 1 x 10 6 por ml vermes em cada frasco. </li><li> Descongelar vermes por incubação dos tubos a 37 ° C durante aproximadamente 2 minutos até quase todo o gelo é derretido. Em uma câmara de fluxo laminar, transferir os vermes para um frasco estéril contendo mCeHR e colocá-los a 20 ° C. </li></ol> 6. Determinar o efeito de Hemin Concentração de Crescimento e Reprodução em mCeHR <ol><li> Use mídia mCeHR axênicos para ensaio de crescimento dependente de nutrientes de C. elegans. Para determinar o efeito of concentração hemina sobre o crescimento e desenvolvimento sem fim, use sincronizado vermes N2 axênicos ou outras cepas de interesse. Sincronizar larvas L1, como descrito acima na seção 4. </li><li> No dia seguinte, pelota e contar larvas L1 sincronizado e diluir a 1 verme por mL de mídia axênicas. Adicione cloreto de hemina a 10 mM em 0,3 M de NH 4 OH e ajustar a pH 8, utilizando HCl concentrado. </li><li> Adicionar 800 ul de meio mCeHR para uma placa de 24 poços. Adicionar 100 vermes para cada poço em 100 ul de meio. Adicionar cloreto de hemina a cada poço em concentrações que variam de 0 uM, 1,5 uM a 1,000 uM em triplicado. Assegurar que todos os poços contêm a mesma concentração de 0,3 M de NH 4 OH. Suavemente ressuspensas os vermes antes pipetando para assegurar que 100 larvas L1 são adicionados a cada poço. </li><li> Examine os vermes diariamente e observe o crescimento em cada concentração em cada poço. Contar o número de vermes após 9 dias de incubação e traçar o número de vermes / ml correspondem a cada hemeconcentração (Figura 1). </li></ol> 7. Efeito da Hemin Concentração em Heme Sensor Worms <ol><li> Incubar sincronizados L1 vermes sensores heme (Phrg-1 :: GFP) em mCeHR contendo 4 pM, 8 pM, 10 uM e 20 uM de hemina. </li><li> Imagem os vermes usando microscopia confocal de fluorescência, 48 pós horas de incubação (Figura 2). </li></ol> 8. Utilizando mCeHR gerar Transgênicos C. elegans Usando Microparticle Bombardeio Nota: O procedimento para a criação e a realização de bombardeamento com micropartículas usando vermes cultivados em mCeHR unc-119 (ed3) é mostrada na figura 3 13. <ol><li> Preparação Worm <ol><li> Transferir cerca de 1 x 10 6 assíncronas unc-119 (ed3 vermes) em 90 ml de meio mCeHR em um balão de 175 centímetros 2 uma semana antes do bombardeamento. Permita que os vermes a crescer a 20 ° C ona plataforma de agitação em ~ 70 rpm (Figura 3-1). Nota: Uma semana mais tarde, a densidade de sem-fim deve ser significativamente maior com diversos vermes adultos grávidos. Aproximadamente 3 x 10 7 vermes será necessária para bombardeamento com micropartículas. </li><li> Frio JM109 E. coli semeadas 10 placas NGM cm no gelo. </li><li> Transferir os vermes axênicos para dois tubos de 50 ml e centrifugar cónicas dos vermes a 800 xg durante 2 min. Aspirar o sobrenadante e ressuspender os vermes em cada tubo de 50 ml de tampão M9 (Figura 3-2). </li><li> Permita que os vermes adultos para sedimentar por gravidade por 10 a 15 min em gelo. Nota: Os sedimentos de 2 ml agora deve conter&gt; 90% vermes adultos. Este sedimento 2 ml devem ter aproximadamente 5 x 10 6 de vermes e é suficiente para um bombardeamento de micropartículas. </li><li> Casaco de toda a superfície da placa de JM109 refrigerados semeadas NGM com o sedimento de 2 ml de vermes. Deixe o líquido ser absorvido completamente em seguida, deixar a placapor 30 min no gelo antes de prosseguir (Figura 3-3). </li></ol></li><li> Preparação de partículas de ouro <ol><li> Pesar 30 mg de partículas de ouro para um tubo de microcentrífuga siliconizada. Adicionar 1 ml de etanol 70% e vortex do tubo durante 5 min. </li><li> Permitir que o tubo em repouso durante 15 min, depois, centrifugar a 6000 xg durante 10 seg para sedimentar as partículas de ouro. Retirar o sobrenadante utilizando uma pipeta, tocando cuidadosamente a ponta para o lado oposto do tubo com as partículas de ouro. </li><li> Lavar as partículas de ouro com 1 ml de água estéril, vortex durante 1 min e centrifugar brevemente o tubo a 6.000 xg durante 10 seg. Repetir o passo de lavagem, duas vezes, em seguida, voltar a suspender as partículas de ouro, em 0,5 ml de glicerol a 50% estéril. Nota: A concentração final das partículas de ouro será de 60 mg / ml e pode ser armazenado durante 1 a 2 meses a 4 ° C. </li></ol></li><li> Preparar a mistura de partículas de DNA a ouro <ol><li> Combinar 10 ug do ADN de plasmídeo desejado com 10 &amp;# 956; g do unc-119 plasmídeo de resgate em uma siliconizada 1,5 ml tubo de microcentrífuga. Adicionar 50 mL de água DI e 100 ul de solução de partículas bem ressuspenso ouro depois vórtice durante 1 min. </li><li> Adicionar 150 ul de 2,5 M CaCl2 e vortex a solução novamente durante 1 min. A esta solução, adicionar 60 ul de espermidina 0,1 M e vortex durante 3 a 5 min. </li><li> Pulso centrifugar a solução a 6.000 xg durante 10 seg, remover o sobrenadante e adicionar 300 ul de etanol a 70%. Vortex bem durante 1 min, centrifugar a 6.000 xg pulso, remove-se o sobrenadante e ressuspender em 170 ul de etanol a 100%. Vortex vigorosamente a solução durante 5 a 10 minutos, em seguida centrifuga pulso e remover o sobrenadante. </li></ol></li><li> Bombardeio (Figura 3-3) Nota: Este protocolo realiza-se com o sistema de entrega de partículas especificado na Tabela de Materiais / equipamento. Siga as instruções para o instrumento de entrega de partículas disponível. <ol> &lt;li&gt; Coloque uma folha de papel de tecido em uma placa de 15 cm. Com uma pinça, mergulhe sete macrocarriers individualmente em etanol 100% e colocá-las sobre o tecido para secar. </li><li> Limpe bem a câmara de biobalística, titular microtransportador, e placa-alvo com etanol 70%. Limpe e autoclave as telas de parada antes de cada procedimento de bombardeio. </li><li> Coloque as macrocarrriers no suporte usando uma pinça e imprensa no suporte usando a ferramenta de estar. </li><li> Uniformemente distribuído 20 ul do DNA de partículas de ouro revestidas, no centro de cada microtransportador e, em seguida, permitir que a solução seque completamente. </li><li> Limpar as duas partes do divisor de pressão com etanol e montar com etanol limpo discos de ruptura. Aperte o divisor de pressão montado e discos de ruptura na câmara de bombardeamento. </li><li> Montar uma tela de parada autoclavado com o titular da microtransportador e coloque o aparelho em cima da prateleira de metal necessárias e trava no lugar. </li><li> Insira o macr montadoAparelho ocarrier na câmara biolística na ranhura colocado abaixo do divisor de tensão e alinhar com o divisor de tensão. </li><li> Tape a placa NGM aberto com os vermes na prateleira placa-alvo e fechar a porta da câmara. </li><li> Para bombardear vermes, ajustar a pressão para que a necessária para quebrar os discos de ruptura. Ligue o vácuo de 28 polegadas de pressão de Hg, em seguida, pressione o botão de fogo até que os discos de ruptura. </li><li> Solte o vácuo e retirar a placa NGM do aparelho. Lave os vermes da placa e redistribuir em vinte placas de 10 cm NGM semeados com JM109 E. coli (Figura 3-4). </li><li> Permitir que os vermes se crescer durante 10 a 14 dias a 25 ° C e as placas de fome. Worms resgatados de defeito unc-119 terá movimento normal e pode ser identificada neste momento. Escolha pelo menos 10 &quot;tipo selvagem&quot; vermes de pratos separados para análise uma vez que estes representam linhagens transgênicas independentes (Figure 3-5). </li></ol></li></ol>

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Os autores declaram que não há concorrentes interesses financeiros ou conflitos de interesses.

Neste protocolo, apresentamos uma mCeHR mídia modificado axênica líquido que permite a rápida C. geração elegans com a produção de um grande número de vermes. Esta mídia mostra várias vantagens como os vermes são cultivados sem contaminar E. coli ou subprodutos de bactérias e pode ser explorada em estudos nutricionais e toxicológicos. O uso de E. coli ou outras bactérias em tais estudos tem vários inconvenientes. Por exemplo, o crescimento das bactérias podem mudar, s...

O nematóide do solo, Caenorhabditis elegans, é um poderoso organismo modelo utilizado em inúmeros estudos da genética à toxicologia. Como resultado do seu tamanho 1 mm de tempo de geração rápida de quatro dias, a facilidade de cultivo, e um grande número da progenitura, estes nemátodos têm sido utilizados num certo número de telas genéticos e farmacológicos 1, 2. Pesquisadores tirar proveito deste worm para identificar moléculas e vias conservadas em sistemas de vertebrados. Estas vias incluem sinais de morte celular, as vias de envelhecimento e metabolismo, e o sistema nervoso 3-6. Além disso, a transparência da C. elegans permite a geração de linhas transgénicas usando repórteres de proteínas fluorescentes, que podem ser visualizadas directamente para analisar os padrões de expressão de genes e localização de proteínas. Em muitos estudos desse nematóide é cultivado em uma superfície à base de agar sólido usando nematóide meio de crescimento placas (NGM) ou lculturas iquid semeada com Escherichia coli como uma fonte de alimento 7,8. Estas fontes alimentares bacterianas podem confundir estudos bioquímicos e toxicológicos com a interferência de bactérias subprodutos que afetam a interpretação dos resultados. A fim de evitar estes efeitos combinados, C. elegans podem ser cultivadas num meio líquido axênicos que é desprovido de bactérias como uma fonte de alimento. Usando essa mídia, cultivadas com sucesso milhões de vermes altamente sincronizadas para muitos padrão C. protocolos elegans, incluindo a análise de microarray de genes diferencialmente regulados em C. elegans expostas a diferentes concentrações de heme e a produção de vermes transgénicas usando bombardeamento de genes. Esta mídia é quimicamente definidos e modificados a partir de uma receita original formulada pelo Dr. Eric Clegg 9. Utilizando este suporte mCeHR, identificámos com sucesso genes envolvidos na homeostase do heme, que se refere a genes que respondem a heme (hrg s) 10, o qual farianão ter sido possível em condições de crescimento normais que utilizam placas de agar semeadas com E. NGM coli. Neste protocolo, descrevemos o procedimento para a introdução e manutenção de C. elegans crescidos em E. coli para o mCeHR axênica e utilizar este método para obter um grande número de vermes para a produção transgénica C. linhas elegans utilizando micropartículas bombardeio. Nós também apresentamos estudos que mostram a utilidade de usar mídia axênicos para determinar a exigência nutricional de C. elegans utilizando heme como um exemplo. Estes estudos mostram que o uso de meios de comunicação mCeHR permite o crescimento rápido de um grande número de C. elegans para muitas aplicações a jusante utilizados por pesquisadores verme.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">MgCl2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">M-2393</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">S-4641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium citrate.H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-1722</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C455-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">M87-100</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnCl2</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">Z-0152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Fe(NH4)2(SO4)2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-1018</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C70-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5 -monophosphate, sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-1752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5 -phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanosine 2 - and3&#160; -monophosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine 5 -phosphate, disodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">U-6375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymine&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T0376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N-Acetylglucosamine</td>
			<td style="width:108px;">Sigma</td>
			<td>A3286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-Alanine</td>
			<td style="width:108px;">Fisher</td>
			<td>S25648&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">p-Aminobenzoic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-9878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Biotin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">B-4639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cyanocobalamine (B-12)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">V-2876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Folinate (Ca)&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-7878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-0761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacinamide</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-3376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantetheine</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-2125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantothenate (Ca)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pteroylglutamic Acid (Folic Acid)</td>
			<td style="width:108px;">ACRCS</td>
			<td style="width:121px;">21663-0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxal 5&#39;-phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-3657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxamine.2HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-9158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6280</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">Riboflavin 5-PO4(Na)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">R-7774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Thiamine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-6,8-Thioctic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1395</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-5379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Choline di-acid citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">myo-Inositol</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">I-5125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-Glucose</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-7520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactalbumin enzymatic hydrolysate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">L-9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain Heart Infusion</td>
			<td style="width:108px;">BD</td>
			<td style="width:121px;">211065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Hemin chloride</td>
			<td style="width:108px;">Frontier Scientific</td>
			<td style="width:121px;">H651-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HEPES, Na salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">H-3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cholesterol</td>
			<td style="width:108px;">J.T. Baker</td>
			<td style="width:121px;">F676-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11140-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Amino Acids Solution</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalidixic acid sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td>N4382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracycline Hydrochloride</td>
			<td style="width:108px;">MP Biomedicals</td>
			<td>2194542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biolistic Delivery System</td>
			<td style="width:108px;">BioRad</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">Gold particles (Au Powder)&#160;</td>
			<td style="width:108px;">&#160;Ferro Electronic Material Systems</td>
			<td style="width:121px;">6420 2504, JZP01010KM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">or</td>
			<td style="width:108px;"></td>
			<td style="width:121px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gold Particles 1.0 &#956;m</td>
			<td style="width:108px;">BioRad</td>
			<td>&#160;165-2263</td>
			<td></td>
		</tr>
	</tbody>
</table>

culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment

Neste protocolo, apresentamos os materiais necessários, bem como o procedimento para a tomada de C modificado. Legans e habituação e de reprodução da mídia (mCeHR). Além disso, as etapas para expor e aclimatação C. elegans crescidos em E. coli para axênica meios líquidos são descritos. Finalmente, experimentos a jusante que utilizam axênica C. elegans ilustrar os benefícios deste procedimento. A capacidade de analisar e determinar C. elegans exigência nutricional foi ilustrada pelo crescimento N2 tipo selvagem vermes em meio líquido axênicos com concentrações variadas de heme. Este procedimento pode ser replicado com outros nutrientes para determinar a concentração óptima para o crescimento e desenvolvimento de sem-fim ou, para determinar os efeitos toxicológicos de tratamentos com drogas. Os efeitos da concentração do heme variadas sobre o crescimento de tipo selvagem vermes foram determinados por meio de observação microscópica qualitativa e quantificando tele número de vermes que cresciam em cada concentração de heme. Além disso, o efeito de concentrações variadas de nutrientes pode ser ensaiada utilizando vermes que expressam sensores fluorescentes que respondem a alterações nos teores de nutrientes de interesse. Além disso, um grande número de vermes foram facilmente produzida para a geração de C. elegans transgénicos utilizando bombardeamento com micropartículas.

Cultivar C. elegans em axênicos ajudas meio líquido na determinação de nutrientes que são necessários por vermes, sem a interferência de metabólitos secundários produzidos por E. coli. Vermes de tipo selvagem N2 aclimatar a mídia mCeHR dentro de três gerações e mostrar um crescimento comparável a vermes cultivadas em placas bacterianas NGM. Na verdade, esses vermes se tornar grávida dentro de 4 dias, em comparação com 3,5 dias para vermes cultivadas em OP50 bactérias. <p class="jo...

Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

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

C. elegans é normalmente cultivadas em placas de agar sólido ou em meio líquido semeadas com E. coli. Para evitar subprodutos bacterianos de confundir os estudos toxicológicos e nutricionais, utilizamos um meio líquido axênico, CEHR, para crescer e sincronizar um grande número de vermes para uma gama de aplicações a jusante.

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

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

culturing-caenorhabditis-elegans-axenic-liquid-media-creation

Research

JoVE Journal

Biology

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

16.8K visualizações.  University of Maryland.  C. elegans é normalmente cultivadas em placas de agar sólido ou em meio líquido semeadas com E. coli. Para evitar subprodutos bacterianos de confundir os estudos toxicológicos e nutricionais, utilizamos um meio líquido axênico, CEHR, para crescer e sincronizar um grande número de vermes para uma gama de aplicações a jusante. 

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

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

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Geração de transgênicos C. elegans Por biobalística Transformação

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Isolamento e In vitro A ativação do Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Lapso de tempo Microscopia de embriogênese inicial em Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: Sincronização e observação

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Extração de prostaglandina e Análise em<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

A cultura de células epiteliais nasais Humano na interface ar líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

A rastreï¿½el<em&gt; In Vivo</em&gt; Ensaio para mitocondrial Modulators Usando Transgenic Bioluminescent<em&gt; Caenorhabditis elegans</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

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 ...