Agradecemos a Chris Webster para a realização dos experimentos preliminares apresentadas na Figura 3 dos resultados representativos e Jason Watts e Chris Webster pelos comentários úteis sobre o manuscrito. O financiamento para este estudo foi fornecido por uma concessão do National Institutes of Health (USA) (R01DK074114) para JLW. Algumas cepas de nematóides utilizados neste trabalho foram fornecidos pelo Centro de Genética Caenorhabditis, que é financiado pelo Escritório de Investigação NIH Programas de Infra-estrutura (P40 OD010440).

Os ácidos graxos poliinsaturados são sensíveis ao calor, luz e oxigênio. Portanto, é preciso ter cuidado ao preparar pratos de suplementação de ácidos graxos de tal forma que os ácidos graxos não estão expostos ao excesso de calor e luz. Meios de NGM contendo 0,1% de Tergitol (NP-40) é esterilizado e parcialmente arrefecida, após o que os sais de sódio de ácido gordo são adicionados com agitação constante. As placas são deixadas a secar no escuro. A absorção de ácidos gordos por C. elegans cultivadas nestas placas podem então ser monitorizado por cromatografia em fase gasosa. 1. Preparação de Ácidos Graxos Supplemented Mídia <ol><li> Medir componentes de mídia em um frasco de tamanho adequado. Por 1 L, adicionar 17 g de Bacto-ágar, 2,5 g de triptona, 3 g de NaCl, 1 mL de 5 mg / ml de colesterol dissolvido em etanol, e 10 ml de 10% de Tergitol dissolvido em água. </li><li> Adicionar 80% do volume final pretendido de água Millipore e autoclave os meios de comunicação, juntamente com os frascos de vidro vazio (igual ao número de diferenteAs concentrações de ácido gordo a ser testado), bem como apropriado provetas. </li><li> Frescos mídia em um banho-maria regulado a 55 ° C. Enquanto a mídia está esfriando preparar a solução estoque de trabalho do sal sódico do ácido graxo por quebrar aberto o frasco de vidro, utilizando-se as precauções de segurança e garantindo partículas de vidro não se misturam com os ácidos graxos. Pesar ácido graxo suficiente para fazer um estoque de trabalho de 100 mm. </li><li> Levar a solução de ácido gordo para uma concentração final de 100 mM com a água purificada. Totalmente dissolvendo o sal de sódio de ácido gordo, tipicamente leva cerca de 20-30 minutos. Purga-se o estoque de trabalho de ácido gordo com de árgon ou de azoto, porque o contacto com um gás inerte, impede a oxidação. Tapar o frasco e armazenar o material a trabalhar, no escuro, até meios de ter arrefecido. </li><li> (Opcional) Se o material de RNAi é desejado, medir a ampicilina e isopropílico β-D-1-tiogalactopiranósido (IPTG) soluções aqui. </li><li> Quando o agar foi arrefecida a 55 ° C adicionar por 1 L: 1ml de 1 M de MgSO4, 1 ml de 1 M de CaCl2, e 25 ml de tampão fosfato (108,3 g de KH 2 PO 4 e 35,6 g de K 2 HPO 4 por 1 L autoclavado). Adicionar o filtro ampicilina esterilizados e soluções IPTG se fazer placas de RNAi. Para todos os tipos de placas, adicionar água estéril para levar o volume do meio final a 1 L. </li><li> Perto de uma chama, a transferência de mídia a uma autoclave e adequadamente dimensionado cilindro graduado e adicione água estéril morna para volume desejado. Mídia de transferência de volta ao frasco inicial e misturar por agitação. </li><li> Perto de uma chama, transferir mídia para um número de garrafas igual ao número de concentrações a serem testadas através da medição com um autoclavado e dimensionado adequadamente cilindro graduado. Manter os meios de comunicação distribuídos em alíquotas como líquido usando um banho de água até que o estoque de trabalho de ácido gordo foi totalmente dissolvida. </li><li> Coloque uma garrafa em uma placa de agitação e mexa até que a mídia é quente ao toque, mas não quente. Certifique-se de deixar-se suficientetempo para mexer na solução estoque de ácidos graxos e despeje placas perante a mídia começa a solidificar. Se placa de agitação tem controle de temperatura, ajuste-o a 55 ° C. </li><li> Diluir o estoque trabalhando ácido graxo 100 mM na mídia para a concentração final desejada. Mídia Agitar até o precipitado branco é, em solução (aproximadamente um minuto). Mídia pode ainda estar um pouco nublado depois. </li><li> (Opcional) Se o material de RNAi é desejado adicionar ampicilina para 0,1 mg / mL e IPTG a 2 mM de concentração final. </li><li> Despeje mídia usando uma ajuda pipeta automática e uma estéril 25 ml pipeta, adicionando 8 ml de meio por placa de 60 mm ou 25 ml de mídia por placa de 100 mm. Repetir a adição de ácido gordo e de placa de derramamento passos para as garrafas restantes. </li><li> Após o ágar se ter solidificado, cobrir as placas com suplemento de ácidos graxos com uma caixa ou armazenar em temperatura ambiente em uma gaveta bem ventilada para proteger da oxidação luz. </li><li> Semente E. coli OP50 em placas de dois dias depois de ter placas secas.Para placas de 60 mm, uma pipeta 300 uL de uma cultura durante a noite OP50 (cultivados a 37 ° C). Se as placas de ARNi foram vertidas, semear com 300 uL de bactérias de RNAi depois as placas foram secas durante quatro dias. Placa tempo de secagem pode precisar de ser ajustado devido a diferenças de umidade em vários ambientes de laboratório. </li><li> Incubar as placas num ambiente escuro a temperatura ambiente, enquanto a camada de bactérias é a secagem. </li></ol> 2. Induzir a destruição das células germinativas por suplementação de ADGL <ol><li> C. elegans cultivadas em placas contendo 0,3 mM de DGLA (20:3 n-6) se tornar estéril devido à destruição das células germinais em ambos os estágios larvais e adultas nemátodos. <ol><li> Prepara-se uma população de larvas L1 sincronizado por tratamento hermafroditas grávidas com solução de hipoclorito alcalino (para uma solução de 10 ml: 0,5 ml de NaOH a 5M, 2,5 ml de lixívia doméstica, e 7 mL de H2O). Agite suavemente os hermafroditas grávidas nesta solução até que o verme adultos dissolver. Os ovos serão preservados, e pode ser sedimentado por centrifugação a baixa velocidade. </li><li> Lavar a preparação de ovo 3 vezes em tampão M9 (3 g de KH 2 PO 4, 6 g de Na 2 HPO 4, 5 g de NaCl, 1 mL de 1 M de MgSO4, H 2 O a 1 L. Adicionar MgSO4 após autoclavagem). Para fornecer oxigénio suficiente, ressuspender os ovos em um tubo de 15 ml de plástico para um volume final de 5 ml de tampão M9 e rocha num agitador durante a noite. </li><li> Larvas L1 devem ser adicionados às placas suplementadas dois dias após a sementeira com OP50, ou um dia após o plaqueamento das bactérias de RNAi. Incubar vermes a 20 ° C durante três dias, ou até se alcançar o estágio adulto. </li></ol></li><li> Os vermes adultos podem ser marcados para a esterilidade, utilizando um microscópio de dissecação com suficientemente alta ampliação para visualizar os embriões em desenvolvimento no útero. Perda de células germinativas de sucesso aparecerá como um vazio útero clara de ovos. </li><li> Worms também pode ser marcado por fixação e coloração com um nucleicocid corante diamindinophenylindole (DAPI), o que facilita a visualização de núcleos de células germinais e de embriões em desenvolvimento. Uma mancha DAPI rápida é conseguida seleccionando vermes em uma gota de tampão M9 sobre um vidro de relógio 9. <ol><li> Flood relógio de vidro com 1 ml de 0,2 ng / m. DAPI em solução de etanol a 95%, deixar repousar durante aproximadamente 5 min. </li><li> Escolha vermes em uma gota de Vectashield em um slide, e depois cobrir com uma lamela. </li><li> Lamela Seal e armazenar a 4 ° C no escuro durante a noite para a coloração ideal, no entanto slides podem também ser vistos imediatamente. Marcar a presença ou ausência de núcleos de células germinativas, utilizando um microscópio de fluorescência equipado com uma lâmpada de UV e um filtro. </li></ol></li></ol> 3. Confirmando Ácidos Graxos Captação por Cromatografia Gasosa Composição geral de ácidos graxos do C. elegans pode ser determinado através da produção de ésteres metílicos de ácidos gordos (FAMEs), que são então separados e quantificados usando gás chromatography 4. <ol><li> Colete 500-1.000 vermes adultos, lavando-as fora das placas com água e transferir para um 13 milímetros x 100 mm parafuso de vidro tubo superior silanizada. </li><li> Vamos resolver vermes pela gravidade, e, em seguida, remover o máximo de água possível com uma pipeta Pasteur de vidro. </li><li> Lave vermes uma vez com água, e depois novamente remover o máximo de água possível. </li><li> FAMEs são formados por adição de 1 ml de 2,5% de H 2 SO 4, em metanol, e, em seguida, o aquecimento a 70 ° C durante 1 hora em um banho de água. </li><li> Retire os tubos do banho-maria e deixe esfriar por 1 min. </li><li> Extrair EMAGs pela adição de 1,5 ml de água e 0,25 ml de hexano. </li><li> Recap tubos e agitar vigorosamente. </li><li> Tubos de centrifugação numa centrífuga clínica de mesa durante 1 min na velocidade máxima para separar o hexano a partir de um solvente aquoso. </li><li> Transferir hexano (camada superior) para uma inserção de GC frasco dentro de um frasco de GC, tomando cuidado para não transferir qualquer da fase aquosa. Para GC umnálise, 1-2 ul de FAMEs em hexano é injectada numa coluna capilar de cromatografia gasosa polar adequado para análise EMAGs. O Agilent 7890 GC injector é de 250 ° C, com uma taxa de fluxo de 1,4 ml / min, e o forno do GC foi programado para uma temperatura inicial de 130 ° C, que é mantida durante 1 min. Subsequentemente, a temperatura é em rampa de 10 ° C / min até 190 ° C, e, em seguida, em rampa novamente a 5 ° C / min até 210 ° C e mantida durante mais 1 min. </li><li> Para assegurar que a absorção de DGLA ocorreu, analisar EMAGs por detecção de ionização de chama (FID) ou espectrometria de massa (MS) de detecção, utilizando-se padrões autênticos para a identificação de C. elegans ácidos graxos. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Aqui, descrevemos um método de suplementação de C. elegans com ácidos gordos insaturados na dieta. Como mencionado acima, é preciso ter cuidado na preparação dos pratos PUFA suplementados porque a natureza reativa das ligações duplas em PUFAs faz com que esses ácidos graxos para ser sensível à oxidação através do calor e da luz 11. Para evitar a oxidação, é importante adicionar o AGPI para o meio de agar depois de os meios de comunicação de líquido tenha arrefecido para 55 ° C e...

Os ácidos gordos são componentes estruturais essenciais das membranas, bem como moléculas de armazenamento de energia eficiente. Além disso, os ácidos gordos podem ser clivada a partir das membranas celulares por lipases e ser enzimaticamente modificada para produzir uma sinalização efectores. De ocorrência natural de ácidos gordos poli-insaturados (PUFAs) contêm duas ou mais ligações duplas cis. Os ômega-3 os ácidos gordos e os ácidos graxos ômega-6 são distinguidos um do outro com base nas posições de ligações duplas em relação ao final de metila do ácido graxo. Dietas saudáveis ​​exigem tanto de ômega-3 os ácidos graxos ômega-6 e. No entanto, as dietas ocidentais são particularmente ricas em ômega-6 ácidos graxos e pobre em ômega-3 ácidos graxos. Uma alta ómega-6 para ácidos gordos ómega rácio-3 está associada com um risco aumentado de doenças cardiovasculares e inflamatórias, no entanto, as funções benéficas e prejudiciais precisas de ácidos gordos específicos não são bem compreendidos 2. Os lombriga elega Caenorhabditisns é útil no estudo de função ácido graxo porque sintetiza todas as enzimas necessárias para produzir uma gama de ácidos graxos ômega-6 e ômega-3, incluindo um omega-3 dessaturase, uma atividade que está ausente na maioria dos animais 3,4. Mutantes sem enzimas dessaturases de ácido gordo não produzem PUFA específicos, que conduz a uma variedade de defeitos do desenvolvimento e neurológicas 4-6. Para estudar os efeitos fisiológicos dos ácidos gordos alimentares, temos desenvolvido um ensaio bioquímico compatível com a análise genética utilizando tanto mutante e RNAi técnicas knock-down em C. elegans. A suplementação com PUFA específicos é conseguida por adição de uma solução de sal de sódio de ácido gordo para o meio de agar antes do vazamento. Isto resulta em PUFA absorção pela E. fonte de alimento coli, onde se acumula nas membranas bacterianas. C. elegans ingerir a bactéria contendo PUFA, e essa suplementação alimentar é suficiente para resgatar a defecst de mutantes PUFA com deficiência. A suplementação da maioria dos ácidos graxos não tem efeitos prejudiciais para os animais selvagens, no entanto, ômega-6 ácidos graxos específicos, especialmente o ácido gama-linolênico dihomo (DGLA, 20:3 n-6) causam uma destruição permanente de C. células germinativas elegans 7,8. Cromatografia em fase gasosa é usado para monitorar a absorção do ácido gordo completada na fonte alimentar bacteriana (ou OP50 ou HT115), bem como nos nemátodes. A adição do Tergitol detergente (NP-40) nos meios permite a distribuição uniforme de ácidos gordos através de toda a placa e uma absorção mais eficiente dos ácidos gordos pela E. coli e os nemátodos. Verificou-se que os ácidos gordos insaturados são facilmente absorvido pelas bactérias e C. elegans, mas a absorção de ácidos gordos saturados, é muito menos eficiente. Este artigo irá descrever passo a passo a forma de complementar o ágar com ácidos graxos, bem como a forma de controlar a absorção de ácidos graxos em poe nematóide utilizando cromatografia gasosa.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

Os ácidos graxos são essenciais para inúmeras funções celulares. Eles servem como moléculas de armazenamento de energia eficientes, compõem o núcleo hidrofóbico das membranas, e participe de várias vias de sinalização. Caenorhabditis elegans sintetiza todas as enzimas necessárias para produzir uma gama de ácidos graxos ômega-6 e ômega-3. Isto, combinado com a anatomia simples e variedade de ferramentas genéticas disponíveis, torná-lo um modelo atraente para estudar a função de ácidos graxos. A fim de investigar as vias genéticas que medeiam os efeitos fisiológicos dos ácidos gordos alimentares, temos desenvolvido um método para complementar a C. elegans dieta com ácidos gordos insaturados. A suplementação é um meio eficaz para alterar a composição de ácidos gordos de vermes e também pode ser usado para recuperar defeitos de mutantes deficientes em ácidos gordos. O nosso método utiliza nematóide de meio de crescimento de ágar (NGM), suplementado com sais acidsodium gordo. Os ácidos graxos nas placas suplementadas se tornar incorporaçãoted nas membranas da fonte alimentar bacteriana, a qual é então feita pela C. elegans que se alimentam de bactérias suplementados. Os requerentes também descrevem um protocolo de cromatografia em fase gasosa para monitorar as alterações na composição de ácidos gordos que ocorrem em vermes suplementados. Esta é uma maneira eficiente para complementar as dietas de grandes e pequenas populações de C. elegans, permitindo uma gama de aplicações para este método.

A suplementação de C. elegans dieta é limitada pela capacidade da fonte de alimentação das bactérias a absorção e incorporação de ácidos gordos na membrana bacteriana. Para determinar a capacidade de E. coli OP50 assimilar vários ácidos gordos nas suas membranas, OP50 foram semeados em meios sem suplemento, 0,1 mM e 0,3 mM, concentrações de ácido esteárico (18:00), oleato de sódio (18:1 n-9), e DGLA de sódio (20 : 3n-6). As placas foram secas à temperatura ambiente durante 2 dias no...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

&lt;em&gt; Caenorhabditis elegan &lt;/ em&gt; é um modelo útil para explorar as funções de ácidos graxos poliinsaturados em desenvolvimento e fisiologia. Este protocolo descreve um método eficiente de complementar o &lt;em&gt; C. elegans &lt;/ em&gt; dieta com ácidos graxos poliinsaturados.

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

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

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K visualizações.  Washington State University.  Caenorhabditis elegan é um modelo útil para explorar as funções de ácidos graxos poliinsaturados em desenvolvimento e fisiologia. Este protocolo descreve um método eficiente de complementar o C. elegans dieta com ácidos graxos poliinsaturados. 

Vídeo: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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 ...

Biologia

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Placa sólida baseada na restrição dietética Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

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, ...

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 ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purificação e Análise de Catadores Elegans Extracelulares

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantificando o declínio proteostático específico do tecido em elegans caenorhabditis

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

Automating Aggregate Quantification in Caenorhabditis elegans

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

Automating Aggregate Quantification in Caenorhabditis elegans

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

Automatizando quantificação agregada em caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...