Name: cultivation of caenorhabditis elegans in three dimensions in the laboratory
Uploaded: 2016-12-12T14:00:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Este trabalho foi apoiado por uma nova Investigator Grant [2014R1A1A1005553] da Fundação Nacional de Pesquisa da Coreia (NRF) para JIL; e uma Universidade Yonsei líder futuro Desafio Grant [2015-22-0133] para JIL

1. Prepare Soluções para NGT-3D e NGB-3D <ol><li> Preparar as seguintes soluções esterilizadas: 1 L de solução de NaCl a 0,1454 M, 1 L de 1 M de CaCl2, 1 L de 1 M de MgSO4, Lisogenia Broth (LB), 1 L de 1 H KPO tampão 4 (108,3 g de KH 2 PO 4 e 35,6 g de K 2 HPO 4, e preencher H2O a 1 L). Produção de NGM usando essas soluções podem ser encontradas em um protocolo anterior 10. </li><li> soluções autoclave a 121 ° C, 15 min. </li><li> Preparar 50 ml de uma solução estéril a 5 mg / ml de colesterol. Em um tubo cónico de 50 mL, misturar 0,25 g de colesterol e 50 ml de 99,99% de etanol e misture bem. Não autoclave. Esterilizar a solução colesterol utilizando uma seringa de 50 ml com um filtro de seringa de 0,45 um. Isso também irá remover qualquer colesterol não dissolvido. </li><li> (Opcional) Preparar 10 ml de uma 150 mM de 2&#39;-desoxi-5-fluorouridina estoque (FUdR). Em um tubo cónico de 15 ml, 0,3693 g misturarde FUdR e 10 ml de água destilada. Agite bem. </li></ol> 2. Prepare Cultura das bactérias para NGT-3D e NGB-3D <ol><li> Inocular 10 ml de caldo LB com bactérias. As bactérias comuns usados para a C. elegans alimentação é a estirpe de E. coli OP50. </li><li> Cultura a inoculação bacteriana a 37 ° C num incubador com agitação durante a noite. </li><li> Na preparação para uma diluição em série, alíquotas de 9 ml da solução de NaCl em 15 ml estéreis tubos cónicos. Por exemplo, alíquotas de 9 ml em um total de 7 tubos para obter uma diluição de 10 -7 estirpe de E. coli OP50. </li><li> Utilizando uma pipeta estéril de 1.000 ul, pipeta de 1 ml da cultura bacteriana ou cultura bacteriana diluída para o novo tubo estéril com 9 ml de solução de NaCl e agitar com vortex a mistura de 10 ml bem. Repita até a diluição bacteriana desejada é atingida. NOTA: A diluição bacteriana desejada depende do tipo e condição de bactérias, bem como as condições experimentais exactos.Por exemplo, um 10 -6 - diluição da estirpe de E. coli OP50 10 -8 era suficiente para produzir a partir de 1 - 200 colónias bacterianas por 6,5 ml tubo NGT-3D. Worms desenvolvido e reproduzidos normalmente quando o número de colónias foi superior a 60, o que ocorreu a uma diluição de 10 -6 fiavelmente - 10 -7. Para NGB-3D, usar uma diluição de 10 -8. </li></ol> 3. Fazer NGT-3D e NGB-3D (200 ml) <ol><li> Depois de preparar a cultura bacteriana, misturar 0,6 g de NaCl, 1 g de agar granulado, e 0,5 g de peptona, em um balão de 500 ml. Inserir uma barra de agitação magnética para o balão. </li><li> Adicione 195 ml de água destilada e cobrir a boca do balão com folha de alumínio. </li><li> Autoclave de 121 ° C durante 15 min. </li><li> Colocar o balão autoclavada quente sobre uma placa de agitação, e agitar a uma velocidade moderada durante pelo menos 2 h. Arrefece-se o balão a 40 ° C. Certifique-se de arrefecer o suficiente como continuar a partir daqui a altas temperaturas pode levar a opacificação do agar acabado.No entanto, as temperaturas mais baixas podem resultar no endurecimento prematuro do agar. <ol><li> (Opcional) Para acelerar o processo de resfriamento, colocar o balão em um 40 ° C banho de água 15 min antes de colocar em uma placa de agitação. </li></ol></li><li> Quando a temperatura do meio de ágar atinge 40 ° C, adicionar 200 ul de 1 M de CaCl2, a 200 ul de 5 mg de solução de colesterol / ml, 200 ul de 1 M de MgSO 4 e 5 ml de 1 M de KPO tampão 4, tal como a solução continua a agitar para concentrações finais de 1 mM de CaCl2, 5 ug / ml de colesterol, MgSO 4 1 mM, KPO4 1 mM. <ol><li> (Opcional) Para o ensaio do tempo de vida NGT-3D adicionar 80 ul de FUdR a 150 mM para uma concentração final de 120 pM 12. </li></ol></li><li> Adicionar 6 ml de 10 ml de cultura bacteriana diluído do passo 2.4 para dentro do frasco directamente. <ol><li> (Opcional) Para NGT-3D, retire 6 ml de ágar antes do passo 3.6 e mantê-lo aquecido separadamente. Este suporte vai ser utilizado para os livre de bactériascamada superior. </li></ol></li><li> Usando procedimentos de esterilização, dispensar a mídia em uma câmara de cultura estéril. Verifique se a tampa da câmara fecha hermeticamente. <ol><li> (Opcional) Para evitar a formação de colónias de bactérias na superfície superior do ágar, fazer uma camada de meio de ágar livre de bactérias a partir do passo 3.6.1 em cima antes que o material a partir do passo 3.7 endurece completamente. Isto irá criar uma zona livre de bactérias no topo da NGT-3D. </li><li> Para NGT-3D, despeje 6,5 ml de meio em 8 ml tubos de ensaio de plástico claras para fazer uma camada de agar de bactérias, e dispensar cuidadosamente 200 ul de meios de comunicação sem bactérias na parte superior do meio de cultura semi-endurecido 3D para fazer uma fina bacteria- camada livre no topo. </li><li> Para NGB-3D, despeje 65 ml de mídia na 25 centímetros garrafa de cultura celular 2 clara de plástico. Esta quantidade deve encher o corpo da garrafa de 25 centímetros de cultura 2 de células. </li></ol></li><li> Deixar câmaras verticalmente à temperatura ambiente durante uma semana para permitir que as colónias bacterianas a crescerpara um tamanho considerável de pelo menos 1 mm de diâmetro. <ol><li> (Opcional) Para o ensaio vida na NGT-3D, câmaras cubra com papel alumínio para evitar a degradação luz do FUdR. </li></ol></li></ol> 4. Medida da aptidão da População Sem-fim na NGT-3D (Brood Relativa Tamanho Assay) <ol><li> Escolha um verme estágio L4 usando um fio de platina pegar e transferência em uma placa NGM sem bactérias. Permitir worm para mover-se livremente ao redor por alguns minutos para remover bactérias aderidas ao corpo. </li><li> Repita o passo 4.1 e certificar-se de que o worm é livre de bactérias. Geralmente, duas repetições de 4.1 são suficientes. </li><li> Com cuidado, coloque o worm limpo na superfície da mídia 3D com uma picareta fio de platina. O worm deve eventualmente entrar no agar agar para a matriz 3D. </li><li> Feche a tampa solta permitindo um pouco de ar para entrar no tubo, mas impedindo a secagem do ágar. </li><li> Incubar durante 96 horas a 20 ° C. </li><li> Após 4 dias, a fechar as tampas hermeticamente e colocar ocâmara de cultura NGT-3D em um banho de água a 88 ° C para fundir o ágar. O calor mata vermes, mas seus corpos permanecem intactos. NOTA: Usando 8 ml tubos para NGT-3D, 20 - 30 minutos de incubação é geralmente suficiente. </li><li> Usando uma pipeta de vidro, transferir os meios derretido sobre de 9 cm de prato de petri de plástico. pipetas de plástico não são aconselhados, como vermes pode muitas vezes ficar com eles. </li><li> Usando um microscópio de dissecação transmissão estéreo, contar o número de vermes no L3, L4, e estágios adultos. Não conte vermes que estão fase L2 e mais jovens, como as gerações F1 e F2 podem ser confundidos aqui. Assim, este ensaio é um ensaio tamanho da ninhada em relação ao invés de um ensaio total de tamanho da ninhada. </li></ol> 5. Imagem e registrar o comportamento Worm na NGB-3D <ol><li> Escolha um verme estágio L4 usando um fio de platina escolher e remover qualquer bactéria coladas à superfície através da transferência a uma placa NGM sem bactérias e permitindo-lhe mover-se livremente para alguns min. </li><li> Repita o passo 5.1 para garantir que o worm é livre de bactérias. </li><li> Cuidadosamente colocar o verme limpo para o centro da superfície de agar do NGB-3D perto do gargalo da garrafa com uma escolha de fio de platina. O worm deve eventualmente entrar no agar agar para a matriz 3D. </li><li> Feche a tampa solta permitindo um pouco de ar para entrar no tubo, evitando a secagem do ágar. </li><li> Imagem ou gravar o worm sob um microscópio de dissecação transmissão estéreo. Ajuste o foco como o worm se move através da matriz 3D. </li></ol>

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The authors have nothing to disclose.

O cultivo laboratorial de C. elegans utilizando as placas de meio de crescimento nematóide clássicos foi crucial para as centenas de descobertas importantes que a pesquisa em C. elegans tem prestado. Aqui, nós apresentamos novos métodos de cultivar C. elegans em um ambiente que reflete com mais precisão seus habitats tridimensionais naturais. Embora outros métodos têm sido utilizados para observar C. elegans em 3D 13, este é o primeiro protocolo que permite a cultur...

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table><tbody><tr><td>LB broth, Miller (Luria-Bertani)</td><td>Difco</td><td>224620</td><td></td></tr><tr><td>Sodium chloride</td><td>DAEJUNG</td><td>7548-4400</td><td>58.44 MW</td></tr><tr><td>Agar, Granulated</td><td>Difco</td><td>214530</td><td></td></tr><tr><td>Peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Calcium chloride, dihydrate</td><td>Bio Basic</td><td>CD0050</td><td>2*H&lt;sub&gt;2&lt;/sub&gt;O; 147.02 MW</td></tr><tr><td>Cholesterol</td><td>Bio Basic</td><td>CD0122</td><td>386.67 MW</td></tr><tr><td>Ethyl alcohol</td><td>B&amp;amp;J</td><td>RP090-1</td><td>99.99%; 46.07 MW</td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Bio Basic</td><td>MN1988</td><td>120.37 MW</td></tr><tr><td>Potassium phosphate, monobasic, anhydrous</td><td>Bio Basic</td><td>PB0445</td><td>136.09 MW</td></tr><tr><td>2&#039;-Deoxy-5-fluorouridine</td><td>Tokyo Chemical Industry</td><td>D2235</td><td>246.19 MW</td></tr><tr><td>Potassium phosphate, dibasic, anhydrous</td><td>Bio Basic</td><td>PB0447</td><td>174.18 MW</td></tr><tr><td>Multi-Purpose Test Tubes</td><td>Stockwell Scientific</td><td>ST.8570</td><td>8 ml</td></tr><tr><td>Test Tube Closures</td><td>Stockwell Scientific</td><td>ST.8575</td><td></td></tr><tr><td>Cell Culture Flask</td><td>SPL Lifescience</td><td>70125</td><td>25 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Research Stereo Microscope</td><td>Nikon</td><td>SMZ18</td><td></td></tr><tr><td>High-Definition Color Camera Head</td><td>Nikon</td><td>DS-Fi2</td><td></td></tr><tr><td>PC-Based Control Unit</td><td>Nikon</td><td>DS-U3</td><td></td></tr><tr><td>NIS-Elements Basic Research, Microscope Imaging Software</td><td>Nikon</td><td>MQS32000</td><td></td></tr></tbody></table>

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.

A construção de NGT-3D é um protocolo simples e directo que resulta em um tubo de ensaio de agar-enchido com pequenas colónias bacterianas espaçadas ao longo do ágar (Figura 1A). Worms pode circular livremente através da matriz de agar, encontrar e consumir as colónias de bactérias. Para confirmar se o C. elegans podem se reproduzir e crescer normalmente na NGT-3D, comparou fertilidade e desenvolvimento larval em 3D com placas padrão 2D NGM. No ensaio ...

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

Nós apresentamos um método simples para a construção de sistemas de cultivo nematóide 3D chamado NGT-3D e NGB-3D. Estas podem ser usadas para estudar a aptidão nematóide e comportamentos em habitats que são mais semelhantes aos naturais Caenorhabditis elegans habitats do que as placas padrão 2D cultura elegans laboratório C..

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 ...

cultivation-caenorhabditis-elegans-three-dimensions

Research

JoVE Journal

Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.8K visualizações.  Yonsei University.  Nós apresentamos um método simples para a construção de sistemas de cultivo nematóide 3D chamado NGT-3D e NGB-3D. Estas podem ser usadas para estudar a aptidão nematóide e comportamentos em habitats que são mais semelhantes aos naturais Caenorhabditis elegans habitats do que as placas padrão 2D cultura elegans laboratório C.. 

Vídeo: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

O nemátode Caenorhabditis Elegans - um modelo versátil na Vivo para estudar interações do anfitrião-micróbio

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genética

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

Baseados na placa de cultivo em grande escala de Caenorhabditis elegans: preparação da amostra para o estudo de alterações metabólicas em Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Bioquímica

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

Microscopia 4D: Desvendando caenorhabditis elegans Desenvolvimento Embrionário usando Microscopia Nomarski

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Biologia do Desenvolvimento

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 ...

Biologia

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Um chip microfluídico simples para crescimento a longo prazo e imagem de Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Bioengenharia

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Um Método para o cultivo embrionário<em&gt; C. elegans</em&gt; Células

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

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

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

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 ...

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

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

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

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

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

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

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

Cultura de Longo Prazo de Caenorhabditis elegans Individuais em Meio Sólido para Monitoramento Longitudinal de Fluorescência e Intervenções Aversivas

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

Cultura de Longa Duração e Monitoramento de Caenorhabditis elegans Isolados em Meio Sólido em Dispositivos Multipoços

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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