Эта работа была поддержана пилот награду от центра Натан Шок (P30 AG13283) и R01 гранта (R01 AG028516) из Национального института старения (NIA) А.-LH

<html> 1. Создание калибровочной кривой для оценки концентрации бактерий В этом разделе описывается метод оценки концентрации бактерий для любого данного штамма бактерий (например, OP50, HT115), который будет использоваться в качестве источника пищи для экспериментов Диетические ограничения на языке C. Элеганс. Отдельные калибровочные кривые должны быть установлены для каждого штамма бактерий используется. <ol><li> Привить единственная колония E. кишечной бактерии OP50 выбрать из свежих полоса бактерий на LB агар в 3 мл жидкого бульона LB. </li><li> Место OP50 культуры в 37 ° C шейкере и позволяют бактериям расти быстро. </li><li> Подготовка серийного 2-кратный разведения бактериальной суспензии из ночной культуры (2x, 4x, 8x, 16x, 32x). </li><li> Измерить абсорбцию (оптической плотности) каждого разведения при 600 нм на спектрофотометре в трех экземплярах. </li><li> Далее разбавленный каждой бактериальной суспензии 1000000 раз (1000-кратного разбавления дважды). </li><li> Распространение 0,4 мл бактериальной суспензии каждого разведения ровно на 100 мм пластины агара LB. </li><li> Место LB пластины с OP50 в 37 ° C инкубаторе в течение 18-24 часов. </li><li> Подсчитайте количество бактериальных колоний, выросших на каждой пластине. Колония насчитывает используются для расчета КОЕ (колониеобразующих единица) на мл для каждого образца. </li><li> Соотношение полученных OD 600 ценности и OP50 бактерий концентрации могут быть созданы путем построения OD 600 в зависимости от концентрации бактерий (КОЕ / мл) и выполняя анализ линейной регрессии (рис. 1). </li></ol> Решения: <table border="1"><tbody><tr><td colspan="2"> Отвар Лурия (LB), 1л: </td></tr><tr><td > 10 г </td><td> Бакто-триптон </td></tr><tr><td> 5 г </td><td> Дрожжевой экстракт </td></tr><tr><td> 10 г </td><td> NaCl </td></tr></tbody></table> Добавить деионизированной воды 1л. Автоклавы и хранить при комнатной температуре. 2. Подготовка твердой пластиной, основанных на пищевых ограничений (SDR) пластин В этом разделе описывается подготовка пластин для использования в твердой пластиной основанных на пищевых ограничений (СДР) экспериментов. Как правило, мы считаем, бактерий концентрации 1 х 10 11 КОЕ / мл, как вволю кормления состоянии и 1 х 10 8 КОЕ / мл в качестве оптимального кормления DR условием для этого метода. Тем не менее, 4-6 различных концентраций бактерий могут быть необходимы для создания полной зависимости от дозы отношений для SDR и его ответы. <ol><li> Привить единственная колония E. Coli OP50 бактерий в 3 мл бульона LB жидкости. </li><li> Место OP50 культуры в 37 ° C шейкере и позволяют бактериям расти в течение 6-8 часов. </li><li> Передача OP50 культуру новых колбу, содержащую 500 мл LB бульоне, и место OP50 культуры еще в 37 ° C шейкер в одночасье. </li><li> Измерить диаметр 600 из OP50 культуры для определения бактерий концентрации (концентрации рассчитывается по калибровочной кривой, генерируемых в часть 1). Соответствующие разведения могут быть необходимы для точного измерения оптической плотности. </li><li> Гранул OP50 бактерий центрифугированием при 1500 г в течение 20 минут. </li><li> Ресуспендируют бактерий к концентрации 1 х 10 12 КОЕ / мл. </li><li> Подготовка 10 мл культуры бактерий в шести различных концентраций: 1 х 10 12, 10 11, 10 10, 10 9, 10 8, 10 7 КОЕ / мл. </li><li> Пипетка 0,2 мл культуры бактерий в центре 60 мм затвердевших NGM пластины. Не прикасайтесь к поверхности пластины с пипетки, а ники на поверхности позволяют червей зарываются в агар пластин. </li><li> Место плит в 37 ° C инкубаторе в течение 1 часа. </li><li> Применяют 10 мкл из 100мг/мл карбенициллин и 10 мкл 50мг/мл канамицин для каждой пластины для ареста рост бактерий. </li><li> Магазин пластины с различными концентрациями антибиотиков убитых бактерий при температуре 4 ° C для дальнейшего использования (до одного месяца). Важно подготовить и хранить достаточно пластин для всего планируемого эксперимента, по крайней мере 1-2 дней вперед. Используя ту же партию пластин в течение всего эксперимента является предпочтительным. </li><li> Чтобы проверить жизнеспособность бактерий, очистить некоторые бактерии из SDR тарелки и разложил ее на пустую тарелку NGM с карбенициллин и канамицина. Место пластины в 37 ° C инкубаторе в течение 6-8 часов, а затем проверить для роста бактерий (нет роста бактерий должны быть соблюдены). </li></ol> Решения <table border="1"><tbody><tr><td colspan="2"> Нематоды Рост Медиа (НГО), 1л: </td></tr><tr><td > 3 г </td><td> NaCl </td></tr><tr><td> 2,5 г </td><td> Бакто-пептон </td></tr><tr><td> 20г </td><td> Агар </td></tr></tbody></table>ove_content &quot;&gt; Автоклав в течение 40 минут и дайте остыть до 65 ° С, затем добавить: <table border="1"><tbody><tr><td > 1 мл </td><td> 1 М MgSO 4 </td></tr><tr><td> 1 мл </td><td> 1 М CaCl 2 </td></tr><tr><td> 1 мл </td><td> 5 мг / мл Холестерин </td></tr><tr><td> 25 мл </td><td> 1М фосфата калия, рН 6,0 </td></tr></tbody></table> Сразу же вылить жидкость NGM на 60 мм х 15 мм чашки Петри в 10 мл аликвоты 3. Настройка SDR экспериментов На протяжении большей части SDR-соответствующих исследований, таких как жизнь анализ диапазона и плодородия профилирования, подготовка возрастных синхронизированы населения червей имеет важное значение. Для других исследованиях, просто перенести червей регулярные OP50 пластины пластины SDR инициировать диетических ограничений. В этом разделе, протокол о создании анализ продолжительности жизни для животных СПЗ описывается как пример. <ol><li> Использование выбора платинового червь для передачи 20-30 репродуктивно активных взрослых на несколько регулярных пластин NGM с OP50. В зависимости от числа яиц, необходимых для экспериментов, более взрослые могут быть необходимы. </li><li> Оставьте пластин при 20 ° С в течение 4 часов, чтобы позволить откладки яиц. </li><li> Удалить взрослых от пластинки. Плиты должны быть визуально проверены на яйца. </li><li> Оставьте пластин при 20 ° С и позволяют потомства, чтобы развиться в Late-L4/day 1 взрослый этап. Обычно это занимает 3 дня для N2 диких червей типа при температуре 20 ° C. </li><li> Передача подходящее число 1-й день взрослым на пластине SDR инициировать диетических ограничений. </li><li> Для анализа продолжительности жизни, создать в общей сложности 6-8 пластин с 10-20 червей на пластине для каждого условия SDR. </li><li> Оценка жизнеспособности каждого червя каждые 2 дня, пока все черви умерли. Очень важно, что черви быть переданы на свежий пластин SDR каждые 12-48 часов, чтобы избежать полного лишения пищи. </li></ol> 4. Представитель Результаты: Показанный на рисунке 1 представлена ​​зависимость кривой представитель калибровки для оценки OP50 концентрации по отношению к OD 600. Уравнение порожденных лайнер регрессионного анализа могут использоваться для всех будущих расчетов OP50 бактерий концентрации. Показанный на рисунке 2 является примером опробование эффект хронического лечения SDR на среднюю продолжительность жизни различных штаммов червя. Синяя линия представляет нормальную дозозависимый ответ дикого типа червей SDR. Красная линия показывает, что влияние продолжительности жизни SDR частично подавлены СРБ-2 э, в то время как DAF-16 (mu86) не оказывает влияния на SDR-индуцированной расширение жизни. Подробности метода для проведения анализа в продолжительности жизни C. Элеганс описана в предыдущей литературе 1-3. <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="Рисунок 1" /> Рисунок 1. Представителю результате калибровочной кривой для оценки Е. Coli OP50 концентрации. OP50 концентрации строится в зависимости от измеряемой величины OD 600. Планки погрешностей = SD Ночь бактериальной культуры разбавляли 1.5x, 2x, 4x, 8x, 16x, 32x до измерения абсорбции, а затем разводят 1000 х дважды, прежде чем измерение КОЕ. <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="Рисунок 2" /> Рисунок 2. Представителю результате C. Элеганс жизни эксперимент сравнения дикого типа N2 животных (синий) с двумя различными мутантами (зеленый и красный) в ответ на SDR (Рисунок из Цзин и соавт. 4). Планки погрешностей = SEM

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Диетические ограничения (DR), как известно, увеличивает продолжительность жизни и замедления наступления различных возрастных заболеваний в широком диапазоне видов, включая млекопитающих, 5-7. Оба экологические и генетические манипуляции были использованы для модели DR на C. Эле?...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии </td></tr><tr><td> Карбенициллин </td><td> Duchefa Biochemie </td><td> C0109.25 </td><td> Растворенного в воде. </td></tr><tr><td> Канамицин </td><td> Roche </td><td> 10106801001 </td><td> Растворенного в воде. </td></tr><tr><td> Бакто-пептонном </td><td> Becton, Dickinson и компании </td><td> DF0118072 </td><td></td></tr><tr><td> Бакто-триптон </td><td> Becton, Dickinson и компании </td><td> DF0123075 </td><td></td></tr><tr><td> Бакто-дрожжевой экстракт </td><td> Becton, Dickinson и компании </td><td> DF0127071 </td><td></td></tr><tr><td> Агар </td><td> Becton, Dickinson и компании </td><td> DF00145170 </td><td></td></tr><tr><td> Стерильная трубка культуры </td><td> США Научная </td><td> 1485-0810 </td><td></td></tr><tr><td> Спектрофотометр </td><td> Amersham Biosciences </td><td> Гена Квант про </td><td> Amersham сейчас является частью компании GE Healthcare. </td></tr><tr><td> Препаровальная лупа </td><td> Zeiss </td><td> Stemi 2000 </td><td></td></tr><tr><td> Центрифуга </td><td> Beckman Coulter </td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

Сокращение потребления пищи без недоедания или голода как известно, увеличивает продолжительность жизни и замедления наступления различных возрастных заболеваний в широком диапазоне видов, в том числе млекопитающих. Это также приводит к снижению массы тела и плодородия, а также более низкие уровни глюкозы в плазме крови, инсулина и IGF-1 у этих животных. Это лечение часто упоминается как диетические ограничения (DR) или ограничение калорийности (CR). Элеганс нематоды Caenorhabditis стала важным организма модель для изучения биологии старения. Оба экологические и генетические манипуляции были использованы для модели DR и показали продлить продолжительность жизни C. Элеганс. Однако многие из сообщили DR исследования в С. Элеганс было сделано путем распространения животных в жидких средах, в то время как большая часть генетических исследований старения поле были сделаны на стандартных твердых агара в чашках Петри. Здесь мы приводим протокол DR с использованием стандартных твердых NGM агар основе пластины с убитых бактерий.

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Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Здесь мы приводим протокол для проведения твердой пластиной, основанных на пищевых ограничений в<em&gt; С. Элеганс</em&gt; С убитых бактерий.

Твердые пластины основе Диетические ограничения в Caenorhabditis Элеганс</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 ...

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Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K Views.  University of Michigan. Здесь мы приводим протокол для проведения твердой пластиной, основанных на пищевых ограничений в С. Элеганс С убитых бактерий.

Video: 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.

Измерительный Caenorhabditis Элеганс Продолжительность жизни на твердых средах

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

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.

Выделение и В пробирке Активация Caenorhabditis Элеганс Сперма

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.

Покадровый Микроскопия раннего эмбриогенеза в Caenorhabditis Элеганс

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.

Основной<em&gt; Caenorhabditis Элеганс</em&gt; Методы синхронизации и наблюдения

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.

Добыча простагландинов и анализа в<em&gt; Caenorhabditis Элеганс</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.

Диетических добавок полиненасыщенных жирных кислот в<em&gt; Caenorhabditis Элеганс</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.

Выращивание<em&gt; Caenorhabditis Элеганс</em&gt; В трех измерениях в лаборатории

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.

Измерение коэффициентов потребления кислорода в неповрежденной Caenorhabditis elegans

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.

Количественная оценка тканеспецифического снижения протеостатического снижения caenorhabditis elegans

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.

Автоматизация количественной оценки агрегатов в 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 ...