Somos gratos a George Jarvis, Martha Bock, e Bette Hecox-Lea, Laboratório de Biologia Marinha, por sua ajuda na filmagem.

1. Preparação de Mídia Nota: Use meio diluída Brujewicz água do mar artificial da salinidade 16,5 ppt (PSU). Outros seawaters artificiais também são frequentemente usados ​​para cultura Brachionus rotíferos 25,26. <ol><li> Adicionar NaCl a 454 mM, 26 mM de MgCl2, 27 mM de MgSO4, 10 mM de KCl e 10 mM de CaCl 2 a 4,5 L de água destilada (volume final será de 5 L). Em alternativa, usar água de diluição desionizada em vez de água destilada. Adicionar CaCl2 depois de se dissolverem todos os outros sais. </li><li> Prepare o M NaHCO solução 0,48 3 estoque (concentração de 200x). Adicionar 25 ml de solução para o acima. A concentração final de NaHCO 3 é de 2,4 mm. </li><li> Preparar a solução stock M NaBr 0,4 (concentração 500x). Adicionar 10 ml da solução para o acima. A concentração final de NaBr é de 0,8 mm. Perfaz-se 5 L com água destilada. </li><li> Filtra-se a solução com um filtro de membrana de 0,45 um.Diluir duas vezes com água estéril antes da sua utilização (v / v). Nota: É possível fazer uma concentração 2x de Brujewicz água do mar artificial como uma solução estoque. </li></ol> 2. Condições Gerais Cultura <ol><li> laboratório-raise Cultura ou rotíferos selvagens-capturado em um copo de 100 ml estéril entre 20 e 30 ° C. As temperaturas mais elevadas resultam em expectativa de vida mais curtos e reprodução acelerada. Usar 25 ° C para a conveniência dos experimentos. A densidade de rotíferos ou o volume de água do mar artificial não é um grande problema aqui. </li><li> Cultura espécies de microalgas alimentares em água do mar artificial 11. Veja Snell et al. (2014) para mais detalhes 11. Tipicamente, usar Tetraselmis tetrathele (~ 2 x 10 5 células / ml) 27,28, T. suecica (~ 6 x 10 5 células / mL), 25,29 e Nannochloropsis oculata (~ 7 X 10 6 células / ml 30,31). Uma vez que dieta algas (espécies, Condit culturaíons, composições bioquímicas) influencia significativamente o tempo de sobrevivência de rotíferos Brachionus, use o mesmo lote de microalga em todos os grupos experimentais. </li><li> Manter uma população de ações de rotíferos em uma cultura em lote através da alimentação e mudando media ocasionalmente (por exemplo, alimentar a cada 2 dias e inocular todas as semanas). Muitos recém-nascidos e adultos portadores de 2-3 ovos pode ser observado quando rotíferos estão em condições óptimas. Nota: Uma forma alternativa de obter rotíferos experimentais é para chocar ovos de repouso. Rotíferos eclodidos de ovos de repouso são considerados para ser bem sincronizados, e o seu tempo de vida não é significativamente diferente da de rotíferos eclodidos de ovos amictic 11. No entanto, rotíferos de ovos que descansam iniciar a reprodução mais cedo do que os de ovos amictic. Assim, é necessária cautela para medições de suas características reprodutivas. </li></ol> 3. Sincronização de rotíferos pelo Pré-cultura <ol><li> Selecione um único rotíferos do stpopulação e cultura ock como descrito no 2,1-2,3 para estabelecer uma sub-população que vai ser utilizado para experiências. Normalmente cultura durante duas semanas. </li><li> Recolha rotíferos portadores de ovos da sub-população (a cobrar o dobro do número de indivíduos que serão utilizados para o experimento). Cultura-los como uma única coorte (densidade: ~ 50 indivíduos / mL) em uma placa de cultura de 6 poços, como descrito no 2,1-2,3 no estado fresco de comunicação sob alimentação ad libitum. Controlando a densidade de população é importante porque o meio condicionado afecta a fisiologia reprodutiva de rotíferos Brachionus 32,33. </li><li> Transferir recém-nascidos nascidos de primeiros ovos dos adultos para meios de cultura recém-preparado. Repita este procedimento ao longo de 2-3 gerações. </li><li> Use neonatos nascidos dentro de um determinado período de tempo (por exemplo, &lt;3 h) para medir o tempo de sobrevivência. Para evitar um possível viés na seleção do único indivíduo, embora muito improvável, verificar a reprodutibilidade usando vários independentes sub-populações. Nota: Como rotíferos normalmente se reproduzem assexuadamente sob esta condição, garantir que não haja homens estão presentes durante todo o experimento. Os machos são menores do que recém-nascidos e, normalmente, se mover mais rápido do que as fêmeas. Fêmeas Mictic têm diferentes tempos de vida das fêmeas amictic sob certas condições 29. </li></ol> 4. Medições de Tempo de Sobrevivência <ol><li> neonatos lugar em placas de plástico (tipicamente de 24 ou 48 poços placas, com cada uma das cavidades contendo 1 ml de água do mar artificial). </li><li> Em intervalos de 24 horas, transferir os rotíferos aos meios de cultura recém-preparados ou em intervalos de 12 horas, se a temperatura mais elevada (acima de 30 ° C ou). Grave o número de filhos e se cada indivíduo está vivo ou morto. Grave o rotíferos como morto quando o movimento dos cílios da corona parou completamente. Nota: Rotíferos muitas vezes atribuem às paredes laterais das cavidades. pipetagem suave da água ajuda a encontrá-los. Se rotíferos não são encontrados ou are acidentalmente danificado pelo pipetagem, gravá-las como &quot;censurado&quot;, não como &quot;morto&quot;. </li><li> Remover neonatos quando rotíferos experimentais estão reproduzindo ativamente. Os recém-nascidos crescem rapidamente e às vezes é difícil distingui-los dos rotíferos experimentais. </li></ol> Análise 5. Os dados <ol><li> Criar a curva de sobrevivência de Kaplan-Meier (Figuras 1 e 2) através da representação gráfica taxa de sobrevivência cumulativa no eixo Y e o tempo no eixo x. Esta é a representação mais comum de dados de sobrevivência. Use o teste de log-rank não paramétrico (também chamado de teste de Mantel-Cox) para comparação estatística de tempo de sobrevivência 34. O teste de log-rank também está incluído em outros pacotes estatísticos padrão, tais como JMP e R. Nota: Não use t-teste ou análise de variância (ANOVA), seguido de comparação múltipla paramétrico Student porque distribuição normal geralmente não é encontrado por dados de sobrevivência 35. Além disso, estes metods não levam indivíduos censurado em conta. Mann-Whitney U Test pode ser usada se não houver dados censurados. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+parental+age+effects+on+the+evolution+of+aging.">The role of parental age effects on the evolution of aging.</a> Evolution. 56 (5), 927-935 (2002).</li></ol>

The authors have nothing to disclose.

O actual protocolo descreve um método para medir o tempo de sobrevivência em rotíferos Brachionus. O passo crítico é a sincronização das condições de rotíferos ao longo de várias gerações. Quando rotíferos experimentais são bem sincronizados, um tipo de curva típica I sobrevivência é observada com muito pouco mortalidade precoce como relatado em vários estudos anteriores 18,24,37,38. desvios-padrão de seu tempo de sobrevivência, portanto, tornam-se menores em comparação com rot...

Rotíferos são microscópicos cosmopolita zooplâncton (&lt;1 mm), que constituem o filo rotifera 1. Eles têm um plano de corpo simples composto por cerca de 1000 células somáticas, bem como um dispositivo de roda-ciliar como característica chamada coroa, que é usado para locomoção e de alimentação. A maioria dos rotíferos pertencem a classes monogononta ou bdelloidea, que contêm cerca de 1.600 e 500 espécies, respectivamente 2. Rotíferos monogononta geralmente têm duas fases reprodução sexuada e assexuada (partenogênese cíclico), enquanto rotíferos bdelloid reproduzir por partenogênese obrigatória 3. É, assim, possível a obtenção de indivíduos geneticamente idênticos rotíferos, o que assegura uma elevada reprodutibilidade em experiências. Além disso, eles têm várias outras vantagens como organismos modelo, tal como um tempo de vida curto, facilidade de cultura, a disponibilidade de dados de sequências genómicas e transcriptomic 4-7, e uma posição filogenética a partir de uma única distanterthropods 8 e nemátodos. Rotíferos são, portanto, prometendo modelos de invertebrados em ecológica, toxicológicas e envelhecimento estudos 9-12. O tempo de sobrevivência sob a exposição ao stress ambiental ou produtos químicos é um parâmetro frequentemente medida nestes campos de pesquisa 13-19. No entanto, é preciso ter cuidado quando se mede o tempo de sobrevivência de rotíferos porque é suscetível às condições ambientais de suas mães. Ou seja, no monogononta Manjavacas rotíferos Brachionus, descendentes do sexo feminino a partir de mães com idade têm uma vida útil mais curta do que os de mães jovens; no entanto, a restrição calórica materna (CR) compensa parcialmente os efeitos deletérios da avançada idade materna 20. Em B. plicatilis, CR materno proporciona longevidade prole, o tempo de sobrevivência muito tempo sob fome e de resistência ao estresse oxidativo alta associada com a expressão aumentada de enzimas antioxidantes 21,22. O efeito da idade maternaTambém tem sido observado em rotíferos bdelloid 23. Portanto, as condições de rotíferos experimentais devem ser cuidadosamente sincronizada ao longo de várias gerações antes das medições do tempo de sobrevivência. Aqui nós fornecemos um protocolo para a medição do tempo de sobrevivência em rotíferos Brachionus seguinte sincronização das condições de cultivo ao longo de várias gerações. Jejum intermitente (IF), uma variação do CR onde rotíferos são alimentados periodicamente, foi aplicado para revelar o efeito de sincronização devido aos efeitos conhecidos de SE na longevidade 22,24.

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			<td>Flat bottom</td>
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			<td style="width:119px;">Version 6.0d</td>
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</table>

measurement of survival time in brachionus rotifers: synchronization of maternal conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

A Figura 1 mostra as curvas de sobrevida representativas de populações mal sincronizados (de duas repetições). Neste experimento, os rotíferos ou eram todos os dias Fed [ad libitum grupo (AL)] ou em dias alternados (IF grupo). A sobrevida média foi de 13 e 18 dias na AL e se os grupos, respectivamente. Embora seja bem sabido que se estende a vida útil do rotíferos, esta experiência não conseguiu detectar uma diferença estatisticamente significat...

Watch this Scientific Journal Video about Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions at JoVE.com

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Measurement of Survival Time in<em&gt; Brachionus</em&gt; Rotifers: Sincronização de condições maternas

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

measurement-survival-time-brachionus-rotifers-synchronization

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Biology

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

8.4K visualizações.  The University of Tokyo. Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Vídeo: Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

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.

Lapso de tempo Microscopia de embriogênese inicial em 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 ...

Biologia

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: Sincronização e observação

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

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

As formulações para liofilização de bactérias e sua influência na sobrevivência das células

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Medição quantitativa de invadopodia mediada proteólise da matriz extracelular em Contextos individuais e multicelulares

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Medição do Tempo de vida em<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Medição dos Níveis de heme em células de mamíferos Síntese

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

A medição da taxa metabólica em<em&gt; Drosophila</em&gt; Usando respirometria

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Baseado-Probe-PCR em tempo real Abordagens para a medição quantitativa de microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Sincronização de<em&gt; Caulobacter crescentus</em&gt; Para Investigação do Ciclo Celular Bacteriana

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

Acompanhamento Temporal de progressão do ciclo celular utilizando citometria de fluxo, sem a necessidade de sincronização

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...