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

이 작품은에 의해 지원되었다 새 탐정 그랜트 [2014R1A1A1005553] JIL에 한국 연구 재단 (NRF)에서; 와 연세대 학교 미래 리더 챌린지 그랜트 [2015-22-0133] JIL에

1. NGT-3D 및 NGB-3D를위한 솔루션을 준비 <ol><li> 다음 멸균 솔루션을 준비 : 0.1454 M NaCl 용액 1 L, 1 M CaCl2를, 1 M 황산 1 L 1 L, 원성 국물 (LB), KH 2의 1 M KPO 4 버퍼 (108.3 g 1 L PO 4 및 K 2 35.6 g HPO 4, H 2 O 기입 L 1). 이러한 솔루션을 사용 NGM의 제조 이전 프로토콜 (10)에서 찾을 수있다. </li><li> 121 ° C, 15 분에서 오토 클레이브 솔루션을 제공합니다. </li><li> 멸균 5 ㎎ / ㎖ 콜레스테롤 용액 50 ㎖를 준비합니다. 한 50㎖ 원추형 튜브에 콜레스테롤의 0.25 g 및 99.99 % 에탄올 50ml를 혼합하고 잘 혼합한다. 오토 클레이브하지 마십시오. 0.45 μm의 주사기 필터로 50 mL를 주사기를 사용하여 콜레스테롤을 살균 용액. 이것은 또한 어떤 용해되지 않은 콜레스테롤을 제거합니다. </li><li> 2&#39;- 데 옥시 -5- fluorouridine (FUdR) 주식의 150 mM의 10 ㎖를 준비합니다 (선택 사항). 15 ml의 원추형 튜브에, 0.3693 g을 혼합FUdR 증류수 10 ㎖의. 잘 흔들어. </li></ol> 2. NGT-3D 및 NGB-3D에 대한 박테리아 문화를 준비합니다 <ol><li> 박테리아 10 ml의 LB 배지에 접종. 공급 C. elegans의 사용되는 표준 박테리아는 대장균 OP50이다. </li><li> 문화 밤새 진탕 배양기에서 37 ° C에서 세균 접종. </li><li> 일련의 희석에 대비, 멸균 15 ML 원뿔 튜브에 NaCl 용액의 나누어지는 9 ml의. 예를 들어, 7 개의 튜브에 분취 9 ml의 OP50 균주 E. 콜라이의 10-7 희석을한다. </li><li> NaCl 용액의 9 ml의 멸균 새로운 튜브에 멸균 1,000 μL 피펫, 피펫 세균 배양 1 ml의 희석 세균 배양를 사용하여 잘 10 ml의 혼합물을 소용돌이. 원하는 박테리아 희석에 도달 할 때까지 반복합니다. 주 : 원하는 박테리아 희석 종류 및 조건 세균뿐만 아니라 정확한 실험 조건에 의존한다.예를 들어, 10-6 - 6.5 ml의 NGT-3D 튜브 당 200 세균성 식민지 - OP50 변형 대장균의 10-8 희석는 1 생산하기에 충분했다. 웜 확실 개발 콜로니 수 × 10-6의 희석에서 발생하는 60 때 정상적으로 재생 - 10-7. NGB-3D를 들어, 10-8의 희석을 사용합니다. </li></ol> 3. 만들기 NGT-3D 및 NGB-3D (200 ㎖) <ol><li> 박테리아 배양을 제조 한 후, 0.6 g의 NaCl 1 g의 과립 한천, 및 500 mL의 플라스크에서, 0.5 g 펩톤 섞는다. 플라스크에 자기 교반 막대를 삽입합니다. </li><li> 195 ml의에게 증류수를 추가하고 알루미늄 호일로 플라스크의 입 커버. </li><li> 15 분 동안 오토 클레이브에서 121 ° C. </li><li> 저어 접시에 뜨거운 멸균 플라스크를 놓고, 적어도 2 시간 동안 적당한 속도로 교반한다. 40 ° C까지 플라스크를 냉각. 완성 된 한천의 흐리게 될 수 있습니다 여기에 고온에서 지속적으로 충분히 냉각해야합니다.그러나, 낮은 온도는 한천의 조기 경화의 원인이 될 수 있습니다. <ol><li> (선택 사항), 냉각 프로세스의 속도를 저어 접시에 배치하기 전에 40 ° C의 물 목욕 15 분에 플라스크를 배치합니다. </li></ol></li><li> 한천 매체의 온도가 40 ℃에 도달하면, 용액은 교반 계속되고, 200 ㎕의 1 M CaCl2를 5 ㎎ / ㎖ 콜레스테롤 용액 200 ㎕를 200 ㎕의 1 M 황산 5 mL의 1 M KPO 4 버퍼를 추가 1 mM의 염화칼슘 2, 5 μg의 / ㎖ 콜레스테롤, 1 mM의 황산, 1 mM의 KPO 4의 최종 농도. <ol><li> NGT-3D 수명 분석을 위해 (선택 사항) 120 μM (12)의 최종 농도 150 mM의 FUdR의 80 μl를 추가합니다. </li></ol></li><li> 직접 플라스크에 단계 2.4에서 박테리아 문화를 희석하여 10 ml의 6 ML을 추가합니다. <ol><li> , NGT-3D (선택 사항) 단계 3.6 전에 한천 미디어의 6 mL로 분리하여 따로 따뜻한 유지. 이 매체는 사용할 수없는 것이다 세균최상층. </li></ol></li><li> 무균 절차를 이용하여, 무균 배양 챔버에 미디어 분배. 확인 챔버의 커버가 단단히 종료합니다. <ol><li> (선택 사항), 한천의 표면에 형성되는 세균성 식민지를 방지 3.7 완전히 경화 단계에서 용지 전에 상단에 단계 3.6.1에서 박테리아가없는 한천 미디어의 층을 확인하십시오. 이 NGT-3D의 상단에 박테리아가없는 영역을 생성합니다. </li><li> NGT-3D를 들어, 박테리아 한천 층을 조심스럽게 얇은 세균 -을 만들기 위해 반경 화 3D 미디어의 상단에있는 박테리아가없는 미디어 200 μl를 분배하는 8 ml의 투명한 플라스틱 시험관에 6.5 ml의 미디어를 부어 상단에 자유 층. </li><li> NGB-3D를 들어, 25cm 2 투명한 플라스틱 세포 배양 병에 미디어 65 ml에 붓는다. 이 금액은 25cm 2 세포 배양 병의 본문을 작성해야합니다. </li></ol></li><li> 박테리아 콜로니가 성장하도록 일주일 수직 상온 챔버 남기기적어도 1mm 직경의 상당한 크기. <ol><li> NGT-3D의 수명 분석을 위해 (선택 사항), 알루미늄 호일 커버 챔버 FUdR의 빛 저하를 방지 할 수있다. </li></ol></li></ol> NGT-3D (상대 브루드 크기 분석)에 대한 웜 인구의 4 측정 휘트니스 <ol><li> 선택과 세균이없는 NGM 접시에 전송 백금 와이어를 사용하여 L4 무대 웜을 선택합니다. 웜은 자유롭게 몸에 부착 된 세균을 제거하는 몇 분 동안 이동할 수 있습니다. </li><li> 단계 4.1를 반복하고 웜이 박테리아에서 무료로 있는지 확인하십시오. 일반적으로, 4.1이 반복 충분하다. </li><li> 조심 백금선 픽업과 3D 매체의 표면에 깨끗한 웜 놓는다. 이 웜은 결국 3D 한천 행렬에 한천을 입력해야합니다. </li><li> 커버 느슨하게 일부 공기가 튜브로 얻을 수 있지만, 한천 매체 건조 방지습니다. </li><li> 20 ° C에서 96 시간 동안 품어. </li><li> 사일 후 단단히 뚜껑을 닫고 두는88 ° C의 물을 욕조에 NGT-3D 문화 챔버는 한천을 녹여합니다. 열은 벌레를 죽이는 그러나 그들의 몸은 그대로 유지됩니다. 참고 : NGT-3D에 8 ㎖의 튜브 사용은 20 - 30 분 배양은 일반적으로 충분합니다. </li><li> 유리 피펫을 사용하여 9cm 플라스틱 페트리 접시에 용융 미디어 옮긴다. 벌레가 자주에 충실 수있는 플라스틱 피펫은 권장되지 않습니다. </li><li> 송신 스테레오 해부 현미경을 사용하여, L3, L4에 웜 및 성체 단계의 개수를 카운트. F1을하고 F2 세대가 여기에 혼동 될 수 있으므로, L2 단계 이하이다 벌레를 계산하지 마십시오. 따라서, 이러한 분석은 상대 브루 크기 분석보다는 전체 브루 크기 분석이다. </li></ol> 5. 이미지 및 기록 웜 동작 NGB-3D에 <ol><li> 백금 와이어를 사용하여 L4 무대 웜 선택 선택하고 박테리아가없는 NGM 판에 전송하고 자유롭게 몇 분 동안 이동할 수 있도록하여 표면에 붙어있는 박테리아를 제거합니다. </li><li> 단계를 반복 5.1 하시다 확인하십시오RM은 박테리아에서 무료입니다. </li><li> 조심스럽게 백금 와이어 픽와 병의 목 근처 NGB-3D의 한천 표면의 중심에 클린 웜을 배치합니다. 이 웜은 결국 3D 한천 행렬에 한천을 입력해야합니다. </li><li> 한천 미디어의 건조를 방지하면서 덮개가 느슨하게, 약간의 공기가 튜브에 들어갈 수 있도록 닫습니다. </li><li> 이미지 또는 전송 스테레오 해부 현미경 벌레를 기록합니다. 웜은 3D 매트릭스를 통해 이동으로 초점을 조절합니다. </li></ol>

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The authors have nothing to disclose.

고전 선충류 성장 미디어 판을 사용하여 선충의 실험 재배가 제공 한 C. elegans의에서 연구 중요한 발견의 수백에 매우 중요했다. 여기, 우리는 더 정확하게 자연 입체 서식지를 반영하는 환경에서 C. 엘레을 육성하기 위해 새로운 방법을 제시한다. 다른 방법 3D 13 C. 엘레을 관찰하기 위해 사용되었지만, 이는 고체 차원 매트릭스 웜의 재배를 허용하는 제 1 프로토?...

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.

NGT도 3d의 구조는 한천 (도 1a)에 걸쳐 이격 된 작은 박테리아 콜로니 한천 채워진 시험관 결과 단순하고 간단한 프로토콜이다. 웜 자유롭게 세균성 식민지를 발견하고 소비, 한천 매트릭스를 통해 이동할 수 있습니다. C. elegans의 재현과 NGT-3D 정상적으로 성장할 수 있는지 여부를 확인하기 위해, 우리는 다산과 표준 2D NGM 플레이트와 3D의 애벌레 개발을 ?...

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

우리는 NGT-3D 및 NGB-3D라는 3D 선충 재배 시스템을 구축하는 간단한 방법을 제시한다. 이러한 표준 2D 실험실 C. elegans의 배양 플레이트보다 자연 예쁜 꼬마 선충 서식지에 더 유사 서식지에서 선충의 체력과 행동을 연구하는 데 사용할 수 있습니다.

의 재배<em&gt; 예쁜 꼬마 선충</em&gt; 실험실에서 세 가지 차원에서

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 조회수.  Yonsei University.  우리는 NGT-3D 및 NGB-3D라는 3D 선충 재배 시스템을 구축하는 간단한 방법을 제시한다. 이러한 표준 2D 실험실 C. elegans의 배양 플레이트보다 자연 예쁜 꼬마 선충 서식지에 더 유사 서식지에서 선충의 체력과 행동을 연구하는 데 사용할 수 있습니다. 

비디오: 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.

선 충 꼬마 선 충-모델을 다양 한 비보에 호스트-미생물 상호작용 연구

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

연구

유전학

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.

꼬마 선 충의 대규모 재배 플레이트 기반: 당뇨병에서 대사 변경의 연구에 대 한 샘플 준비

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

생화학

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.

4D 현미경 검사: 노마르스키 현미경을 이용한 예쁜꼬마 선 충 배아 발달 풀기

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

발생학

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.

기본<em&gt; Caenorhabditis elegans</em&gt; 방법 : 동기화 및 전망

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

생물학

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.

Caenorhabditis elegans의 장기 성장 및 이미징을위한 간단한 미세 유체 칩

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

생체공학

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.

배아 배양하는 방법<em&gt; C. 엘레</em&gt; 셀

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.

배양<em&gt; 예쁜 꼬마 선충</em &gt; 미세 폭격에 의한 무균 액체 미디어 및 형질 전환 웜의 생성에

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.

예쁜꼬마선충의 세포구조와 세포세포형태를 연구하기 위한 정량적 접근법

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.

Longitudinal Fluorescence Monitoring and Averseive Interventions를 위한 고체 배지에서 개별 Caenorhabditis elegans 의 장기 배양

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.

Multi-well 장치의 고체 배지에서 분리된 Caenorhabditis elegans의 장기 배양 및 모니터링Long-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, ...

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