Os autores gostariam de agradecer a junho Middleton para o fornecimento de E. cepas faecalis e de orientação com a cultura de bactérias. C. elegans foram fornecidos pela CGC, que é financiado pelo NIH Escritório de Programas de Infra-estruturas de Investigação (P40 OD010440).

1) Elaboração de Nematóides Crescimento Mídia (NGM) <ol><li> Para fazer com que as placas 50, adicionar 1,5 g de NaCl, 8,5 g Agar ultrapura, e 1,25 g de peptona de um 1 Lflask. </li><li> Adicionar 487,5 ml de dH 2 O para o balão. Agite bem para misturar e cobrir abertura do frasco com um pedaço de papel alumínio. </li><li> Esterilizar a solução por tratamento em autoclave, em condições normalizadas (121 psi, 120 ° C, 20 min.) </li><li> Deixar a solução arrefecer até 45 ° C num banho de água. </li><li> Para a solução, adicionar o seguinte na ordem: 500 ul de colesterol a 5 mg / ml de solução (dissolvido em etanol), 500 ul de 1 M de CaCl2, 500 ul de 1 M de MgSO4, e 12,5 ml de tampão KPO4 (54,15 g KH 2 PO 4 e 17,8 g de K 2 HPO 4 em 500 ml de dH2O), tal como descrito em 18. </li><li> Usando pipetas esterilizadas, adicione 10 ml de solução de agar para cada 60 milímetros de poliestireno placa de Petri. Permitir que os meios de comunicação para solidificar à temperatura ambiente overnight. </li></ol> 2) Preparação de B Caldo E. Mídia coli <ol><li> Para fazer com que cinco culturas de 100 ml, adicionar 5,0 g de Bacto-triptona, 2,5 g de extracto de levedura, 5,0 g de NaCl para uma proveta. </li><li> Adicionar água destilada estéril para um volume final de 500 ml. Aquece-se a solução sobre uma placa quente, com agitação, até que os solutos se dissolveram. </li><li> Colocar 100 ml de caldo de B em cinco frascos de vidro (pelo menos, 200 ml de volume). </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Permitir B caldo de arrefecer até à temperatura ambiente antes do uso. </li></ol> 3) Sementeira C. elegans Placas de Manutenção <ol><li> Depois de permitir que as placas NGM a secar à temperatura ambiente durante pelo menos uma semana, inocular 100 ml de um caldo de cultura B com algumas colónias de E. coli (OP50). </li><li> Crescer E. coli, a cultura a 37 ° C durante a noite. </li><li> Pipeta uma ou duas gotas (aproximadamente 10081; l) OP50 de cultura para o centro de cada placa NGM. As placas são geralmente empilhados do lado da tampa para cima. Quando pipetagem (seeding), começam por pipetagem a cultura na placa, na parte inferior da pilha. Trabalhe seu caminho até cada pilha. Tente não mover as placas após a sementeira, porque é importante que a cultura não fique muito perto dos bordos da placa. </li><li> Permitir que as placas semeadas a secar à temperatura ambiente durante pelo menos uma semana antes da utilização. </li><li> Placas semeadas são armazenados (invertido), em recipientes de plástico vedados à temperatura ambiente. </li></ol> 4) A manutenção C. elegans Cepas <ol><li> Para manter bem alimentado C. estoques elegans, use um verme escolher para passar três L4s ou adultos grávidas para um novo, sem sementes NGM placa a cada 3-4 dias. </li><li> Placas de Loja (invertido), de preferência dentro de uma incubadora a 15 - 22 º C. Culturas em ensaios descritos foram armazenadas a 20 ° C. C. elegans desenvolvimento é dependente da temperatura. Como maintenance a temperatura aumenta, o tempo entre os estágios de desenvolvimento diminui. </li></ol> 5) Preparação de Tryptic Soy Agar (TSA), E. faecalis Meios de Cultura <ol><li> Para fazer 50 placas, meça 500 ml de água destilada estéril para um frasco e leve para ferver, agitando, numa placa de aquecimento. </li><li> Adicionar 20 g de pó de ágar TSA ao balão e permita que a solução para dissolver. </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Deixar a solução arrefecer até 45 ° C num banho de água. </li><li> Usando pipetas esterilizadas, adicione 10 ml de solução de agar para cada 60 milímetros de poliestireno placa de Petri. Permitir que os meios de comunicação para solidificar à temperatura ambiente durante a noite. </li></ol> 6) Preparação de Tryptic Soy Broth (TSB), E. faecalis Meios de Cultura <ol><li> Para fazer com que os tubos 250 de cultura, medida a 500 ml de água destilada estéril para um frasco e quente numa placa de aquecimento com agitação. </li&gt; <li> Adicionar 15 g de pó de ágar TSB para o frasco e para permitir que o pó se dissolver. </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Deixa-se arrefecer até à temperatura ambiente, em seguida, pipeta de 2 ml, em tubos de ensaio de 5 ml esterilizadas. </li></ol> 7) Preparação de Heart Infusion (BHI) Mídia Cérebro, com estreptomicina, para E. faecalis Cultura <ol><li> Medir 500 ml de água destilada estéril para um frasco e leve para ferver, agitando, numa placa de aquecimento. </li><li> Adicionar 26 g de meio BHI ao balão e permitir que a solução para dissolver. </li><li> Esterilizar a solução por autoclavagem, em condições normalizadas (121 psi, 120 ° C, 20 minutos). </li><li> Deixa-se arrefecer a 45 ° C num banho de água. </li><li> Utilizando uma técnica estéril, adicionar 0,5 mL de uma solução de estoque de 10 mg de estreptomicina / ml e agite para misturar. </li><li> Adicionar 10 ml da solução a cada 60 milímetros de Petri de poliestireno. </li><li&gt; Permitir mídia para solidificar à temperatura ambiente durante a noite. Placas de Loja (invertida) a 4 ° C até algumas horas antes do uso. </li></ol> 8) Mantendo E. faecalis Cepas <ol><li> Para manter E. estoques faecalis, use uma agulha esterilizada para raia colônias individuais em ágar placas de soja trípticos separados (TSA). </li><li> Crescer as culturas a 37 ° C durante a noite. Placas de Loja (invertida) em um saco ou caixa selada à temperatura ambiente durante várias semanas. </li></ol> 9) Preparar E. Placas faecalis para EIW Assay <ol><li> Inocular uma cultura ml 2 TSB com uma colônia de E. faecalis e incubar durante a noite a 37 ° C. </li><li> Pipetar 20 ul de E. faecalis cultura para o centro de uma placa BHI-estreptomicina preparada, e incubar durante a noite a 37 ° C. </li></ol> 10) Preparar E. Placas coli para EIW Assay (placas de controlo) <ol><li> Inocular um ml B-bro 100º cultura com uma colônia de E. coli OP50 e incubar durante a noite a 37 ° C </li><li> Pipetar 20 ul da cultura OP50 para o centro de uma placa NGM unseeded e permitir que as placas semeadas a secar à temperatura ambiente durante a noite. </li></ol> 11) Ovo em Ensaio Worm <ol><li> Escolha 15-20 encenou-L4 C. elegans (Figura 1) para o centro de uma camada de bactérias numa preparada BHI-Strep-E. placa faecalis. Tenha cuidado para não transferir um monte de OP50 a placa BHI. Escolha o mesmo número de L4s a um controle de placa NGM-OP50. </li><li> Incubar as placas durante 40 horas a 20 ° C. </li><li> Preparar a solução de água sanitária a 20%. Misture a água sanitária comercial (hipoclorito de sódio a 6,0%) com o volume adequado de água destilada. </li><li> Adicione 10 gotas mL de solução de água sanitária para dez locais distintos em uma tampa de plástico (de um worm ou placa bacteriana). </li><li> Usando uma picareta verme, transferir um verme em cada gota de água sanitária. Agite suavemente a ponta dopegar na gota de água sanitária para certificar-se o worm tem lavado o fim da escolha (confirmar vendo a gota sob um microscópio de dissecação). </li><li> Permitir que a cutícula para dissolver durante cerca de 10 min ou até que os vermes se abriu, expelindo os ovos (Figura 2). Tenha cuidado para não transferir os ovos a partir da placa. </li><li> Usando um microscópio de dissecação, a contagem dos ovos em cada gota de água sanitária. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Os passos mais importantes na realização com êxito neste ensaio são os seguintes: 1) usando stocks bem alimentados de C. elegans, 2) cultura de tipos individuais de bactérias na placa de ensaio, 3) identificação precisão encenado L4 vermes para a exposição a E. faecalis, 4), mantendo o tempo de exposição a E. faecalis consistentes em todos os ensaios e 5) branqueamento tempo não deve exceder 10 minutos para impedir a desintegração do ovo. Para este e...

Caenorhabditis elegans, um microscópico, lombriga de vida livre, é um organismo modelo tradicionalmente usado para estudar os processos de desenvolvimento e de sinalização celular por causa de sua anatomia transparente, o desenvolvimento bem caracterizada, totalmente sequenciado o genoma, em tempo de geração curto, e homologia genética de seres humanos . Mais recentemente, C. elegans tornou-se um organismo modelo no campo da toxicologia ambiental e imunidade inata 10, 11. Esses vermes hermafroditas auto-fertilização se tornam sexualmente maduros dentro de dois a três dias de incubação do ovo. Durante o seu ciclo de vida, C. elegans passa por quatro estágios larvais (L1-L4), antes de atingir a idade adulta. Um isolado hermafrodita pode produzir, em média, 300 filhotes dentro de três dias de pico de fecundidade. Em reprodutivamente madura C. elegans hermafroditas, os ovos fertilizados são retidos no interior do útero durante várias horas antes de ser colocada. Onúmero normal de ovos armazenados no útero em qualquer momento (durante o pico da fecundidade) situa-se entre dez e quinze 12. O número de ovos no útero é uma função de ambos, a taxa de produção de ovos e a taxa de postura de ovos. Os ovos fertilizados são expulsos do útero pela contração da musculatura dezasseis vulvares dispostas em torno da abertura da vulva 13. Neurónios motores específicos do hermafrodita (do HSN) e neurónios motores VC sinapse para os músculos da vulva que afetam a contração muscular e, assim, postura comportamento 5,7,13,14. Expulsão dos ovos do útero ocorre devido à atividade coordenada de neurônios e músculos. Culturas de laboratório de C. elegans normalmente são levantadas em uma dieta de não patogênicas de Escherichia coli OP50. No ambiente natural, C. elegans entrar em contacto com uma variedade de fontes de alimentos, tais como as bactérias patogénicas, que podem ser potencialmente prejudiciais. Quando expostos a substâncias nocivas emambiente, C. elegans reter ovos até que o ambiente torna-se mais favorável. Presumivelmente, esta retenção de ovos é um esforço para proteger sua prole. Neste verme ovo em ensaio (EIW), C. elegans estão expostos às bactérias potencialmente patogénicas, Enterococcus faecalis, que é encontrada no meio ambiente. A exposição a formas patogénicas de E. faecalis pode causar infecção intestinal persistente e até mesmo a morte em C. elegans 15. A exposição a outras formas de bactérias patogénicas têm demonstrado afectar a retenção de ovo 16,17, no entanto, o efeito não foi quantificado. Além disso, o efeito de suavemente estirpes patogénicas de E. faecalis, as estirpes que não são imediatamente letal, sobre o comportamento de postura não foi estudado. EIW ensaios envolvem a contagem do número de ovos retidos no útero de C. elegans 4. Embora C. eleganssão transparentes, os ovos que se acumulam no útero pode ser difícil de quantificar num animal intacto. O ensaio EIW envolve branqueamento gravídico adulto C. elegans que foram expostos a bactérias, por um período fixo de tempo. A solução de água sanitária dissolve a cutícula externa deixando os ovos para trás. Os ovos são de refracção para os efeitos de branqueamento, devido à presença de uma casca de ovo. Após o branqueamento, é muito facilmente capaz de contar o número de ovos libertados do útero dos vermes após o branqueamento. O ensaio é descrito um método simples, barato e rápido para quantificar o número de óvulos no útero de uma só vez, e, assim, quantificar os efeitos de E. faecalis sobre retenção de ovo. Este ensaio pode ser utilizado para quantificar o efeito de outros tipos de bactérias, toxinas ou medicamentos ambientais sobre a retenção de ovo. Este ensaio tem também o potencial de ser usada como uma tela para patogenicidade.

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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agar, ultrapure</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">10906</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Peptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Tryptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Brain Heart Infusion dehydrated medium</td>
			<td style="width:191px;">Carolina Biological Supply</td>
			<td style="width:119px;">781781</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">C. elegans, N2 strain</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">Cholesterol</td>
			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:119px;">A11470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Culture plates for C. elegans</td>
			<td style="width:191px;">Tritech Research Inc.</td>
			<td style="width:119px;">T3308</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Culture plates for E. faecalis</td>
			<td style="width:191px;">Fisher Scientific-Fisherbrand</td>
			<td style="width:119px;">875713</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">E. coli (OP50)</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">E. faecalis strains</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>provided by J. Middleton. All isolates were confirmed as enterococci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>
			all strains grew at 44.5 &#186;C and were catalase negative and hydrolyzed esculin. A simplified&#160;dichotomous key based on pigmentation and fermentation reactions for six sugars&#160;(arabinose, mannitol, methyl-&#945;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&#160;presumptive identification of all E. faecalis strains (Efs lacks pigmentation and is arabinose, MGP&#160;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&#160;were confirmed using the API 20 STREP system (Biomerieux).
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microscope</td>
			<td style="width:191px;">Motic</td>
			<td style="width:119px;">SMZ 168B</td>
			<td>any microscope with transmitted illumination and 50X magnification should be sufficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Streptomycin sulfate</td>
			<td style="width:191px;">Fisher BioReagents</td>
			<td style="width:119px;">BP910-50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">236950</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Yeast extract</td>
			<td style="width:191px;">Acros</td>
			<td style="width:119px;">61180-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

C. elegans comportamento de postura de ovos é afetado por estímulos ambientais, como a osmolaridade 1 e vibração 2. Na ausência total de alimentos C. elegans também deixará postura de ovos e manter ovos fertilizados em sua 3 útero. No entanto, o efeito de diferentes fontes de alimentos, especialmente bactérias patogénicas e, particularmente, Enterococcus faecalis, sobre o comportamento de postura não está bem caracterizado. O ensaio in ovo sem-fim (EIW) é uma ferramenta útil para quantificar os efeitos de diferentes tipos de bactérias, neste caso E. faecalis, sobre o comportamento de postura. EIW ensaios envolvem a contagem do número de ovos retidos no útero de C. elegans 4. O ensaio EIW envolve branqueamento encenado, grávida adulto C. elegans para remover a cutícula e separar os ovos retidos do animal. Antes do branqueamento, os vermes são expostos a bactérias (ou qualquer tipo de sinalização ambiental) Por um período fixo de tempo. Após o branqueamento, é muito facilmente capaz de contar o número de ovos retidos no interior do útero dos vermes. Neste ensaio, a um aumento na retenção quantificável de ovos após E. exposição faecalis pode ser facilmente medido. O ensaio EIW é um ensaio comportamental que poderia ser utilizado para rastrear para bactérias potencialmente patogénicos ou a presença de toxinas ambientais. Além disso, o ensaio EIW pode ser uma ferramenta para o rastreio de fármacos que afectam a sinalização de neurotransmissores, pois o comportamento de postura é modulada por neurotransmissores, tais como serotonina e acetilcolina 5-9.

Este ensaio permite a quantificação do número de ovos retidos no interior C. elegans após exposição a E. faecalis. L4 encenado vermes (caracterizados por espaço aberto transparente acima de sua vulva, Figura 1) foram expostos a E. faecalis até a idade adulta. Após 40 horas de exposição a E. faecalis, branqueamento foi realizado. À medida que a cutícula desintegrado na gotícula de lixívia, os ovos se tornou mais evidente (Figura 2). O n?...

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Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Um ensaio ovo-in-worm (EIW) é um método útil para quantificar o comportamento de postura. Alterações na postura de ovos pode ser uma resposta comportamental do organismo modelo<em&gt; Caenorhabditis elegans</em&gt; A substâncias ambientais potencialmente prejudiciais, tais como os produzidos por bactérias patogénicas.

Medição dos Efeitos de bactérias em<em&gt; C. Elegans</em&gt; Comportamento Usando um Retenção Ensaio Egg

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

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16.9K visualizações.  Fairleigh Dickinson University. Um ensaio ovo-in-worm (EIW) é um método útil para quantificar o comportamento de postura. Alterações na postura de ovos pode ser uma resposta comportamental do organismo modelo Caenorhabditis elegans A substâncias ambientais potencialmente prejudiciais, tais como os produzidos por bactérias patogénicas.

Vídeo: Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Geração de transgênicos Estável C. elegans Usando microinjeção

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Biologia

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 ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imagem C. elegans Os embriões utilizando um microscópio de epifluorescência e Software de Código Aberto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

Visualização da função respiratória mitocondrial usando Citocromo C Desidrogenase Oxidase / Succinato (COX / SDH) Histoquímica rotulagem dupla

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Criando ambientes definidos gasosos para estudar os efeitos da hipóxia na<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Um ensaio de explante para avaliar o comportamento celular do mesênquima craniana

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Anticorpo de coloração em<em&gt; C. Elegans</em&gt; Usando o &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Em larga escala Knockdown Gene em<em&gt; C. elegans</em&gt; Usando bibliotecas de alimentação dsRNA para gerar fenótipos robustos perda de função

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Um modelo de anóxia-inanição por isquemia / reperfusão em<em&gt; C. elegans</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...