Siamo grati a George Jarvis, Martha Bock, e Bette Hecox-Lea, Marine Biological Laboratory, per il loro aiuto in riprese.

1. Preparazione di media Nota: utilizzare metà diluita Brujewicz acqua di mare artificiale di salinità 16,5 ppt (PSU). Altri seawaters artificiali sono anche spesso utilizzate per cultura Brachionus rotiferi 25,26. <ol><li> Aggiungere 454 mM NaCl, 26 mM MgCl 2, 27 mM MgSO 4, 10 mM KCl, e 10 mM CaCl 2 al 4,5 l di acqua distillata (volume finale sarà di 5 L). In alternativa, utilizzare acqua di diluizione deionizzata invece di acqua distillata. Aggiungere CaCl 2 dopo aver sciolto tutti gli altri sali. </li><li> Preparare la M NaHCO soluzione 0.48 3 stock (concentrazione 200x). Aggiungere 25 ml di esso alla soluzione precedente. La concentrazione finale di NaHCO 3 è di 2,4 mM. </li><li> Preparare la soluzione di riserva M NaBr 0,4 (concentrazione 500x). Aggiungere 10 ml di esso alla soluzione precedente. La concentrazione finale di NaBr è 0,8 mM. Portare a 5 L con acqua distillata. </li><li> Filtrare la soluzione con un filtro a membrana da 0,45 micron.Diluire due volte con acqua sterile prima dell&#39;uso (v / v). Nota: E &#39;possibile effettuare una concentrazione 2x di Brujewicz acqua di mare artificiale come una soluzione madre. </li></ol> 2. Condizioni generali Culture <ol><li> Cultura laboratorio rilievo o rotiferi selvatici catturati in una sterile bicchiere da 100 ml tra 20 e 30 ° C. Temperature più elevate si traducono in durate di vita più brevi e la riproduzione accelerata. Utilizzare 25 ° C per la comodità di esperimenti. La densità rotifero o il volume di acqua di mare artificiale non è un grosso problema qui. </li><li> Cultura specie di microalghe alimentari in acqua di mare artificiale 11. Vedere Snell et al. (2014) per i dettagli 11. In genere, usare Tetraselmis tetrathele (~ 2 x 10 5 cellule / ml 27,28), T. suecica (~ 6 x 10 5 cellule / ml 25,29), e Nannochloropsis oculata (~ 7 x 10 6 cellule / ml 30,31). Dal momento che le alghe dieta (specie, la cultura Conditioni, composizioni biochimiche) influenza in modo significativo il tempo di sopravvivenza dei rotiferi Brachionus, utilizzare lo stesso lotto di microalga in tutti i gruppi sperimentali. </li><li> Mantenere una popolazione magazzino di rotiferi in una cultura lotto alimentando e cambio di un supporto di tanto in tanto (ad esempio, da mangiare ogni 2 giorni e inoculare ogni settimana). Molti neonati e adulti che portano 2-3 uova possono essere osservati quando rotiferi sono in condizioni ottimali. Nota: Un modo alternativo per ottenere rotiferi sperimentali è a covare le uova di riposo. Rotiferi nati dalle uova di riposo sono considerati essere ben sincronizzati, e la loro durata di vita non è significativamente diversa da quella dei rotiferi nati da uova amictic 11. Tuttavia, rotiferi da uova di riposo iniziare la riproduzione prima di quelli da uova amictic. Quindi, cautela è necessaria per la misura delle loro caratteristiche riproduttive. </li></ol> 3. La sincronizzazione dei rotiferi da Pre-cultura <ol><li> Selezionare una singola rotifero dalla stock popolazione e cultura come descritto in 2.1-2.3 per stabilire una sottopopolazione che verrà utilizzato per esperimenti. In genere la cultura per due settimane. </li><li> Raccogliere rotiferi uova recanti dalla sotto-popolazione (raccogliere un numero doppio di persone che verranno utilizzati per l&#39;esperimento). Li cultura come singola coorte (densità: ~ 50 individui / ml) in una piastra di coltura a 6 così come descritto in 2.1-2.3 in un fresco supporto sotto alimentazione ad libitum. Controllare la densità di popolazione è importante perché il mezzo condizionato incide sulla fisiologia della riproduzione Brachionus rotiferi 32,33. </li><li> Trasferire i neonati nati da uova prima degli adulti a terreni di coltura appena preparata. Ripetere questa procedura per 2-3 generazioni. </li><li> Utilizzare neonati nati entro un certo periodo di tempo (ad esempio, &lt;3 hr) per la misura del tempo di sopravvivenza. Per evitare possibili distorsioni nella scelta del singolo individuo, anche se molto improbabile, controllare la riproducibilità utilizzando diversi independenti sottopopolazioni. Nota: Come rotiferi solito riproducono solo asessuata in questa condizione, garantire l&#39;assenza di maschi sono presenti in tutto l&#39;esperimento. I maschi sono più piccoli di neonati e in genere si muovono più velocemente rispetto alle femmine. Femmine Mictic hanno differenti durate di vita da femmine amictic a determinate condizioni 29. </li></ol> 4. Le misurazioni del Tempo di sopravvivenza <ol><li> Luogo neonati in piatti di plastica (in genere 24 o 48 pozzetti, con ogni ben contenente 1 ml di acqua di mare artificiale). </li><li> A intervalli hr 24, trasferire i rotiferi per terreni di coltura appena preparati o ad intervalli di 12 h se a temperatura elevata (30 ° C o superiore). Registrare il numero di figli e se ogni individuo è vivo o morto. Registrare il rotifero come morto quando il movimento delle ciglia della corona ha completamente fermato. Nota: Rotiferi spesso attaccano alle pareti laterali dei pozzi. pipettaggio delicato dell&#39;acqua aiuta a trovarli. Se rotiferi non si trovano o are danneggiato accidentalmente dal pipettaggio, registrarli come &quot;censurato&quot;, non come &quot;morto&quot;. </li><li> Rimuovere i neonati quando rotiferi sperimentali sono attivamente riprodursi. I neonati crescono rapidamente ed è a volte difficile distinguerli dai rotiferi sperimentali. </li></ol> Analisi 5. I dati <ol><li> Creare la curva di sopravvivenza di Kaplan-Meier (figure 1 e 2) riportando tasso di sopravvivenza cumulativo sull&#39;asse Y e tempo sull&#39;asse X. Questa è la rappresentazione più comune di dati di sopravvivenza. Utilizzare il log-rank test non parametrico (chiamato anche test di Mantel-Cox) per il confronto statistico del tempo di sopravvivenza 34. Il log-rank test è incluso anche in altri pacchetti statistici standard come JMP e R. Nota: non utilizzare t-test o analisi della varianza (ANOVA) seguita da parametrico confronto multiplo di Student distribuzione normale perché di solito non è soddisfatta da dati di sopravvivenza 35. Inoltre, questi methods non prendono gli individui Censored in considerazione. Mann-Whitney U test può essere utilizzato se non ci sono dati censurati. </li></ol>

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The authors have nothing to disclose.

L&#39;attuale protocollo descrive un metodo per misurare il tempo di sopravvivenza in rotiferi Brachionus. Il passo fondamentale è la sincronizzazione delle condizioni di rotiferi per diverse generazioni. Quando rotiferi sperimentali sono ben sincronizzati, una curva di sopravvivenza che tipo tipico è osservata con molto poco mortalità precoce come riportato in diversi studi precedenti 18,24,37,38. Le deviazioni standard del loro tempo di sopravvivenza di conseguenza diventano più piccoli rispett...

Rotiferi sono microscopici cosmopolita zooplancton (&lt;1 mm), che costituiscono il phylum Rotifera 1. Hanno un semplice piano corpo composto di circa 1.000 cellule somatiche, nonché un apparato ciliare ruota-come caratteristica denominata corona, che viene utilizzato per la locomozione e l&#39;alimentazione. La maggior parte dei rotiferi appartengono a classi Monogononta o Bdelloidea, che contengono circa 1.600 e 500 specie, rispettivamente 2. Rotiferi Monogonont hanno generalmente entrambe le fasi riproduttivo sessuale e asessuale (partenogenesi ciclico), mentre rotiferi bdelloid si riproducono per partenogenesi obbligatoria 3. È così possibile ottenere individui rotifere geneticamente identici, che assicura un&#39;elevata riproducibilità in esperimenti. Inoltre, essi hanno diversi altri vantaggi come organismi modello, come una vita breve, facilità di coltura, la disponibilità dei dati di sequenza genomica e trascrittomica 4-7, ed una posizione filogenetica unica distante da unarthropods e nematodi 8. Rotiferi sono quindi promettenti modelli di invertebrati in ecologico, tossicologico, e l&#39;invecchiamento studi 9-12. Il tempo di sopravvivenza in condizioni di esposizione a stress ambientale o sostanze chimiche è un parametro spesso misurato in questi campi di ricerca 13-19. Tuttavia, la cautela è necessaria quando si misura il tempo di sopravvivenza dei rotiferi, perché è suscettibile a condizioni ambientali delle loro madri. Vale a dire, nel monogonont Manjavacas rotifero Brachionus, prole femminile da madri di età hanno una durata più breve rispetto a quelli da giovani madri; tuttavia, la restrizione calorica materna (CR) compensa in parte gli effetti deleteri di avanzata età materna 20. In B. plicatilis, materna CR fornisce longevità prole, il tempo di sopravvivenza a lungo sotto la fame, ed elevata resistenza stress ossidativo associato con una maggiore espressione di enzimi antiossidanti 21,22. L&#39;effetto dell&#39;età maternaè stato osservato anche in rotiferi bdelloid 23. Pertanto, le condizioni di rotiferi sperimentali devono essere attentamente sincronizzate su più generazioni prima di misurazioni del tempo di sopravvivenza. Qui forniamo un protocollo per la misurazione del tempo di sopravvivenza in rotiferi Brachionus dopo la sincronizzazione delle condizioni di coltura per diverse generazioni. Digiuno intermittente (IF), una variazione di CR dove rotiferi vengono alimentati periodicamente, è stato applicato per rivelare l&#39;effetto della sincronizzazione a causa degli effetti ben noti di IF sulla longevità 22,24.

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			<td height="21" style="height:21px;width:268px;">Sodium chloride</td>
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			<td height="21" style="height:21px;width:268px;">Potassium chloride</td>
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			<td height="21" style="height:21px;width:268px;">Sodium bicarbonate</td>
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			<td height="21" style="height:21px;width:268px;">Sodium bromide</td>
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			<td height="21" style="height:21px;width:268px;">Membrane filter (0.45 &#181;m pore size)</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:119px;">HAWP04700</td>
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			<td height="21" style="height:21px;width:268px;">Culture plate, 6-well, non-treated</td>
			<td style="width:135px;">Thomas Scientific</td>
			<td style="width:119px;">6902D01</td>
			<td>Flat bottom</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Culture plate, 48-well, non-treated</td>
			<td style="width:135px;">Thomas Scientific</td>
			<td style="width:119px;">6902D07</td>
			<td>Flat bottom</td>
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			<td height="42" style="height:42px;width:268px;">Tetraselmis, Living</td>
			<td style="width:135px;">Carolina Biological Supply Company</td>
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			<td height="21" style="height:21px;width:268px;">PRISM 6</td>
			<td style="width:135px;">GraphPad Software</td>
			<td style="width:119px;">Version 6.0d</td>
			<td></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.

La figura 1 mostra le curve rappresentative di sopravvivenza delle popolazioni mal sincronizzati (su due repliche). In questo esperimento, rotiferi erano o tutti i giorni della Fed [ad libitum (AL) gruppo] o ogni altro giorno (se il gruppo). La sopravvivenza mediana è stata di 13 e 18 giorni nel AL e se i gruppi, rispettivamente. Sebbene sia ben noto che se prolunga la vita della rotifero, questo esperimento non ha rilevato una differenza statisticamente ...

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.

Misurazione del Tempo di sopravvivenza in<em&gt; Brachionus</em&gt; Rotiferi: Sincronizzazione di materni Condizioni

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

Research

JoVE Journal

Biology

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

8.4K Views.  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.

Video: 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.

Microscopia time-lapse di Embriogenesi All&#39;inizio del 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 ...

Ricerca

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.

Di base<em&gt; Caenorhabditis elegans</em&gt; Metodi: sincronizzazione e di osservazione

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.

Formulazioni per la liofilizzazione di batteri e la loro influenza sulla sopravvivenza cellulare

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.

Misurazione quantitativa della Invadopodia-mediata proteolisi della matrice extracellulare in singoli contesti e pluricellulari

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.

Misura della durata della vita in<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.

La misurazione dei livelli Heme sintesi in cellule di mammifero

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.

Misurazione del tasso metabolico in<em&gt; Drosophila</em&gt; Utilizzando 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.

Base-Probe Real-time PCR Approcci per la misurazione quantitativa dei microRNA

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.

Sincronizzazione di<em&gt; Caulobacter crescentus</em&gt; Per Indagine del ciclo cellulare batterica

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.

Monitoraggio temporale del ciclo cellulare progressione citometria a flusso, senza la necessità di sincronizzazione

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