이 작품은 A. - LH에 네이슨 쇼크 센터 (P30 AG13283)과 노화에있는 국립 연구소 (NIA)에서 R01 그랜트 (R01 AG028516)에서 파일럿 수상에 의해 지원되었다

<html> 1. 세균 농도의 추정을위한 보정 곡선을 수립 이 섹션에서는 C로식이 제한 실험을위한 식품 소스로 사용되며 특정 박테리아 스트레인 (예 : OP50, HT115)의 세균 농도의 추정을위한 방법을 설명합니다 엘레간스. 별도의 교정 곡선은 응력이 사용 각각의 세균에 대해 설립해야합니다. <ol><li> E.의 단일 식민지의 예방 대장균 박테리아 OP50 3 ML 액체 LB의 국물에 LB 한천에 박테리아의 새로운 행진에서 골랐어요. </li><li> 37 장소 OP50 문화 박테리아가 하룻밤 사이에 성장 ° C 뿌리와이 가능하다는 특징을 가지고있다. </li><li> (2X, 4X, 8X, 16X, 32x) 야간 문화에서 세균성 정지 시리얼 2 배 dilutions를 준비합니다. </li><li> 세중의에서 분광 광도계로 600 nm의 각 희석의 흡광도를 (광학 밀도) 측정. </li><li> 또한 각각의 박테리아 현탁액 백만 배 (1000 배 희석 배) 희석. </li><li> 100mm의 LB 한천 플레이트에 균등하게 각 희석의 세균성 정지 0.4 ML을 확산. </li><li> 18~24시간에 대해 37 ° C 배양기에서 OP50와 장소 LB 번호판을. </li><li> 각 접시에 박테리아 식민지 성장의 개수를 계산합니다. 식민지 카운트는 각 샘플에 대해 ML 당 cfu을 (콜로니 형성 단위)을 계산하는 데 사용됩니다. </li><li> 측정된 OD 600 값을 OP50 세균 농도 사이의 관계는 세균의 농도 (cfu / ML)의 함수로 OD 600하려하고 선형 회귀 분석 (그림 1)을 수행하여 설치하실 수 있습니다. </li></ol> 솔루션 : <table border="1"><tbody><tr><td colspan="2"> Luria 맛을 (LB), 1L : </td></tr><tr><td > 10g </td><td> Bacto - tryptone </td></tr><tr><td> 5g </td><td> 효모 추출 </td></tr><tr><td> 10g </td><td> NaCl </td></tr></tbody></table> 1L에 탈이온수를 추가합니다. 압력솥과 실온에서 저장합니다. 2. 고체 판 기반식이 제한 (SDR) 접시의 준비 이 섹션은 고체 판을 기반으로식이 제한 (SDR) 실험에 사용하기 위해 접시의 준비에 대해 설명합니다. 일반적으로, 우리는 고려의 박테리아 농도 1 X 10 11 cfu / ML 광고 libitum 먹이 조건 1과 같은 X이 방법에 대한 최적의 DR 먹이 조건으로 10 8 cfu / ML. 그러나, 4-6 다른 박테리아의 농도는 SDR과 응답을위한 완벽한 복용량 종속적인 관계를 확립해야 할 수도 있습니다. <ol><li> E.의 단일 식민지의 예방 3 ML 액체 LB의 국물에 대장균 박테리아 OP50. </li><li> 37 ° C 통에 OP50 문화를 삽입하여 박테리아가 6~8시간에 대한 성장 수 있습니다. </li><li> 500 ML의 LB의 국물을 포함하는 새로운 플라스크에 OP50 문화를 전송하고, ° C 쉐이크 하루 37 다시 OP50 문화를 놓으십시오. </li><li> 박테리아 농도 (농도가 파트 1에서 생성된 교정 곡선을 사용하여 계산)를 확인하기 위해 OP50 문화의 OD 600 측정. 적절한 dilutions는 흡광도의 정확한 측정에 필요한 수 있습니다. </li><li> 20 분 1,500그램에서 원심 분리하여 펠렛 OP50 박테리아. </li><li> 1 X 10 12 cfu / ML의 농도에 박테리아를 Resuspend. </li><li> 여섯 가지 농도 10 ML 박테리아 문화를 준비 : 1 × 10 12 10 11 10 10 10 9 10 8 10 7 cfu / ML. </li><li> 60mm의 중심에 박테리아 문화의 피펫 0.2 ML은 NGM 접시를 확정. 표면 닉스는 한천 플레이트에 버로우에 벌레를 허용 등 피펫 팁과 접시의 표면을 만지지 마십시오. </li><li> 1 시간 동안 37 ° C 배양기에서 플레이스 번호판입니다. </li><li> 박테리아의 성장을 체포하기 위해 각 플레이트에 100mg/mL carbenicillin과 50mg/mL kanamycin 10 μL의 10μL을 적용합니다. </li><li> 4 항생제 - 살해 박테리아의 다른 농도 ° C 나중에 사용하기 위해 (최대 한 달). 함께 저장 플레이트 앞으로 적어도 1-2일 전체 계획의 실험을위한 충분한 접시를 준비하고 저장하는 것이 중요합니다. 전체 실험 접시 같은 배치를 이용하는 것은 바람직합니다. </li><li> 박테리아의 생존을 확인하려면, SDR 번호판의 일부 박테리아를 긁어과 carbenicillin과 kanamycin과 빈 NGM 접시에 확산. 6~8시간에 대해 37 ° C 배양기에 플레이트를 삽입하고 박테리아의 성장 (어떤 박테리아의 성장이 관찰되지 않을 것입니다)를 확인하십시오. </li></ol> 솔루션 <table border="1"><tbody><tr><td colspan="2"> 선충류 성장 미디어 (NGM), 1L : </td></tr><tr><td > 3g </td><td> NaCl </td></tr><tr><td> 2.5 g </td><td> Bacto - 펩톤 </td></tr><tr><td> 20g </td><td> 한천 </td></tr></tbody></table>ove_content가 &quot;&gt; 40 분 압력솥과 65 식지 ° C, 다음 추가 : <table border="1"><tbody><tr><td > 1 ML </td><td> 1 M MgSO 4 </td></tr><tr><td> 1 ML </td><td> 1 M CaCl 2 </td></tr><tr><td> 1 ML </td><td> 5 MG / ML 콜레스테롤 </td></tr><tr><td> 25 ML </td><td> 1M 칼륨 나트륨, 산도 6.0 </td></tr></tbody></table> 즉시 10 ML의 aliquots의 60mm X 15mm 배양 접시에 액체 NGM를 따르다 3. SDR 실험 설정 이러한 수명 분석 및 불임 프로 파일링, 웜의 시대 - 동기 인구의 준비로 SDR 관련 연구의 대부분 필수적입니다. 다른 연구, 간단하게 식사 제한을 시작하는 SDR 판에 일반 OP50 접시에서 벌레를 전송합니다. 이 섹션에서는 SDR 동물에 대한 수명 분석을 설정하는 프로토콜은 예제로 설명되어 있습니다. <ol><li> OP50 여러 일반 NGM 플레이트에 20-30 reproductively 활성 성인 전송하는 플래티넘 웜 선택기를 사용하십시오. 실험에 필요한 난자의 숫자에 따라 더 많은 성인이 필요할 수 있습니다. </li><li> ° C 4 시간 동안 달걀 누워 수 있도록 20 접시를 남겨주세요. </li><li> 접시에서 어른을 제거합니다. 플레이트는 시각 달걀에 대한 검사를해야합니다. </li><li> 20 접시를 남겨 ° C와 자손이 Late-L4/day 어른 1 단계로 발전 수 있습니다. 보통이 20 N2 야생 타입 웜 3 일이 소요 ° C. </li><li> 식이 제한을 시작하는 SDR 플레이트에 1 일 성인의 적절한 숫자를 전송합니다. </li><li> 수명 분석을 위해, 각 SDR 상태에 대한 번호판 10-20 벌레와 6-8 접시 총을 설정합니다. </li><li> 모든 벌레가 죽었을 때까지 모든 이일 각 웜의 생존을 점수. 그것은 벌레가 음식의 완전한 박탈을 피하​​기 위해 모든 12-48시간 신선한 SDR 플레이트로 전송하는 것이 중요합니다. </li></ol> 4. 대표 결과 : OD 600 관련 OP50 농도의 추정을위한 대표적인 보정 곡선의 줄거리는 그림 1에 표시됩니다. 라이너 회귀 분석에 의해 생성된 방정식은 OP50 세균 농도 이후의 모든 계산을 위해 사용될 수 있습니다. 그림 2에 표시된 것은 다른 웜 변종의 의미 수명에 만성 SDR 트리 트먼트의 효과를 시금의 예입니다. 파란색 라인은 SDR로 야생 타입의 웜의 보통사람들 종속적인 반응을 나타냅니다. DAF - 16 (mu86)는 SDR 유발 수명 연장에 아무런 영향을 미치지 않습니다 동안 빨간색 선은, SDR의 수명 효과가 부분적으로 drr - 2 오에 의해 억압을 나타냅니다. C.의 수명 분석을 수행하기위한 세부 방법 엘레간스은 이전 문학 1-3에 설명되어 있습니다. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="그림 1" /> 그림 1. E.의 추정을위한 보정 곡선의 대표 결과 대장균 OP50 농도. OP50 농도가 측정된 OD 600 값을 함수로 꾸몄다 있습니다. 오차 막대 = SD 숙박 세균성 문화 흡광도 측정하기 전에, 4X, 2X, 1.5x 배속, 16 배속, 32x를 희석하고, 추가로 희석 1,000 X 번 cfu 측정하기 전에했습니다. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="그림 2" /> 그림 2. C. 대표 결과 엘레간스 수명 실험은 (칭 외에서 그림 4.) SDR에 대한 응답으로 두 개의 서로 다른 돌연변이 (녹색과 빨간색)와 야생 유형 N2 동물 (파란색)을 비교. 오류 바 = SEM

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관심 없음 충돌 선언하지 않습니다.

식이 제한 (DR)는 수명을 증가시키고 5-7 포유 동물을 포함한 동물의 다양한 다양한 연령과 관련된 질병의 발병을 지연하는 것으로 알려져 있습니다. 환경과 유전 모두 조작은 C로 모델 DR에 사용되었습니다 세미 정의 액체 매질 8,9, 액체 배지에서 세균성 10-12 음식 소스의 희석하고, 느린 속도 13 행동 돌연변이 해당 피드의 사용 culturing 웜을 포함한 엘레간?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> Carbenicillin </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> 물속에 녹아있는. </td></tr><tr><td> Kanamycin </td><td> 로체 </td><td> 10106801001 </td><td> 물속에 녹아있는. </td></tr><tr><td> Bacto - 펩톤 </td><td> 백톤, 디킨슨 및 회사 </td><td> DF0118072 </td><td></td></tr><tr><td> Bacto - tryptone </td><td> 백톤, 디킨슨 및 회사 </td><td> DF0123075 </td><td></td></tr><tr><td> Bacto - 효모 추출물 </td><td> 백톤, 디킨슨 및 회사 </td><td> DF0127071 </td><td></td></tr><tr><td> 한천 </td><td> 백톤, 디킨슨 및 회사 </td><td> DF00145170 </td><td></td></tr><tr><td> 무균 배양 튜브 </td><td> 미국 과학 </td><td> 1485-0810 </td><td></td></tr><tr><td> 분광 광도계 </td><td> 애머스햄 Biosciences </td><td> 진 퀀트 프로 </td><td> 아머샴 지금은 GE 헬스케어의 일부입니다. </td></tr><tr><td> 해부 현미경 </td><td> 자이스 혈구 </td><td> Stemi 2000 </td><td></td></tr><tr><td> 원심 분리기 </td><td> 베크맨 쿨터 </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

영양 부족이나 기아없이 음식 섭취의 감소가 수명을 증가시키고 포유 동물을 포함한 동물의 광범위한 다양한 연령과 관련된 질병의 발병을 지연하는 것으로 알려져 있습니다. 그것은 또한 체중과 불임의 감소뿐만 아니라 플라즈마 혈당, 인슐린, 이러한 동물의 IGF - 1의 낮은 수준을 초래합니다. 이 치료는 종종식이 제한 (DR) 또는 열량 제한 (CR)이라고합니다. 선충류의 Caenorhabditis의 엘레간스은 노화의 생물학을 공부에 대한 중요한 모델 생물로 등장했다. 환경과 유전 모두 조작이 모델 DR에 사용되고 있으며 C로 수명을 연장 보였습니다 엘레간스. 그러나, C로 보고된 DR 연구의 많은 엘레간스은 노화 분야에서 유전자 연구의 대부분은 배양 접시에서 표준 고체 한천에서 수행하는 동안, 액체 미디어 동물 전파에 의해 수행되었다. 여기 우리는 죽은 박테리아 표준 고체 NGM 한천 기반 판을 사용하여 DR 프로토콜을 제시한다.

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

여기에 단단한 판 기반식이 제한을 수행하는 프로토콜을 제시<em&gt; C. 엘레간스</em&gt; 죽은 박테리아와 함께.

솔리드 플레이트 기반식이 제한에 Caenorhabditis 엘레간스</em

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

solid-plate-based-dietary-restriction-in-caenorhabditis-elegans

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JoVE Journal

Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K 조회수.  University of Michigan. 여기에 단단한 판 기반식이 제한을 수행하는 프로토콜을 제시 C. 엘레간스 죽은 박테리아와 함께.

비디오: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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.

측정 Caenorhabditis 엘레간스</em솔리드 미디어> 수명

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

연구

생물학

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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

분리 및 체외에서</em의> 활성화 Caenorhabditis 엘레간스 정자

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

상순 Embryogenesis의 시간 경과 현미경 Caenorhabditis 엘레간스

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

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 ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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

에서 프로스타글란딘 추출 및 분석<em&gt; Caenorhabditis의 엘레</em

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

에있는 고도 불포화 지방산의식이 보충제<em&gt; 예쁜 꼬마 선충</em

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

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 ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

그대로 꼬마 선 충 에 산소 소비 속도의 측정

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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

Caenorhabditis 예인단에서 조직 특정 전립선 성 감소를 정량화

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

Automating Aggregate Quantification in Caenorhabditis elegans

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

Automating Aggregate Quantification in Caenorhabditis elegans

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

Caenorhabditis elegans의 집계 정량화 자동화

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