Wir danken George Jarvis, Martha Bock und Bette Hecox-Lea, Marine Biological Laboratory, für ihre Hilfe bei der Verfilmung.

1. Herstellung von Medien Hinweis: Verwenden Sie Halb verdünnt Brujewicz künstlichem Meerwasser Salzgehalt 16,5 ppt (PSU). Andere künstliche seawaters werden auch häufig verwendet , um Kultur Brachionus rotifers 25,26. <ol><li> Hinzufügen 454 mM NaCl, 26 mM MgCl 2, 27 mM MgSO 4, 10 mM KCl, und 10 mM CaCl 2 bis 4,5 L destilliertem Wasser (Endvolumen 5 wird L sein). Alternativ dazu können Sie entsalztem Verdünnungswasser anstelle von destilliertem Wasser. In CaCl 2 , nachdem alle anderen Salze aufzulösen. </li><li> Bereiten Sie die 0,48 M NaHCO 3 - Stammlösung (200x Konzentration). 25 ml davon zu der obigen Lösung. Die Endkonzentration von NaHCO 3 beträgt 2,4 mM. </li><li> Bereiten Sie die 0,4 M NaBr-Stammlösung (500x Konzentration). 10 ml davon zu der obigen Lösung. Die Endkonzentration von NaBr 0,8 mM. Machen Sie bis zu 5 L mit destilliertem Wasser. </li><li> Filtern der Lösung mit einem 0,45 um-Membranfilter.Verdünnen zweimal mit sterilem Wasser vor der Verwendung (v / v). Hinweis: Es ist möglich, eine 2x Konzentration von Brujewicz künstlichem Meerwasser als Stammlösung zu machen. </li></ol> 2. Allgemeine Kulturbedingungen <ol><li> Kultur im Labor erhöht oder Wild gefangen Rotatorien in einem sterilen 100-ml-Becher zwischen 20 und 30 ° C. Höhere Temperaturen führen zu kürzeren Lebensdauer und eine beschleunigte Wiedergabe. Verwenden 25 ° C für die Bequemlichkeit der Experimente. Die rotifer Dichte oder Volumen von künstlichem Meerwasser ist kein großes Problem hier. </li><li> Kultur Nahrungs Mikroalge Spezies in künstlichem Meerwasser 11. Siehe Snell et al. (2014) für weitere Einzelheiten 11. Typischerweise verwenden Tetraselmis tetrathele (~ 2 x 10 5 Zellen / ml 27,28), T. suecica (~ 6 x 10 5 Zellen / ml 25,29) und Nannochloropsis oculata (~ 7 x 10 6 Zellen / ml 30,31). Da Diätalgen (Arten, Kultur conditIonen, biochemische Zusammensetzungen) , beeinflusst maßgeblich die Überlebenszeit von Brachionus Rotatorien, die gleiche Menge Mikroalge in allen Versuchsgruppen verwenden. </li><li> Halten Sie eine Stammpopulation von Rotatorien in einer Batch - Kultur durch die Fütterung und wechselnden Medien gelegentlich (zB füttern alle 2 Tage und impfen jede Woche). Viele Neugeborene und Erwachsene 2-3 Eier tragen, können beobachtet werden, wenn Rotatorien unter optimalen Bedingungen sind. Hinweis: Eine alternative Möglichkeit, experimentelle Rotatorien zu erhalten, ist Dauereier auszubrüten. Rotifers aus ruhenden Eier ausgebrütet werden als gut synchronisiert werden, und ihre Lebensdauer ist nicht signifikant verschieden von der aus amictic Eier 11 schraffiert Rotatorien. Allerdings rotifers aus ruhenden Eier beginnen Reproduktion früher als die aus amictic Eier. Daher ist Vorsicht für Messungen ihrer Reproduktionsmerkmale erforderlich. </li></ol> 3. Synchronisation von Rotifers von Pre-Kultur <ol><li> Wählen Sie einen einzelnen rotifer aus dem stock Bevölkerung und Kultur wie in 2,1-2,3 beschrieben eine Subpopulation zu etablieren, die für Experimente verwendet werden. Typischerweise Kultur für zwei Wochen. </li><li> Sammeln egg tragenden Rotatorien vom Subpopulation (eine doppelte Anzahl von Personen zu sammeln, die für das Experiment verwendet wird). Kultur als eine einzige Kohorte (Dichte: ~ 50 Personen / ml) in einer 6-Well - Kulturplatte wie in 2,1-2,3 in einem frischen Medien unter Ad - libitum - Fütterung beschrieben. Controlling Bevölkerungsdichte ist wichtig , weil das konditionierte Medium 32,33 Fortpflanzungsphysiologie von Brachionus rotifers beeinflusst. </li><li> Übertragen ausgebrütet Neugeborenen von der ersten Eier &quot;Erwachsene neu vorbereitet Kulturmedien. Wiederholen Sie diese Prozedur über 2-3 Generationen. </li><li> Verwenden innerhalb eines bestimmten Zeitraums schraffierten Neugeborenen (zB &lt;3 h) , um die Überlebenszeit gemessen wird . Um zu vermeiden, eine mögliche Verzerrung des einzelnen Individuums bei der Auswahl, wenn auch sehr unwahrscheinlich, überprüfen Reproduzierbarkeit unter Verwendung mehrerer independent Subpopulationen. Hinweis: Da Rotatorien der Regel nur ungeschlechtlich unter dieser Bedingung zu reproduzieren, sicherzustellen, dass keine Männchen während des gesamten Experiments vorhanden sind. Die Männchen sind kleiner als Neugeborene und in der Regel bewegen sich schneller als Frauen. Mictic Weibchen haben unterschiedliche Lebensdauern von amictic Weibchen unter bestimmten Bedingungen 29. </li></ol> 4. Messungen der Überlebenszeit <ol><li> Ort Neugeborenen in Kunststoffplatten (typischerweise 24- oder 48-Well-Platten, wobei jede Vertiefung mit 1 ml künstlichem Meerwasser). </li><li> Nach 24 h Intervallen, übertragen Sie die Rotatorien neu vorbereitet Kulturmedien oder in 12 Stunden Intervallen, wenn bei höherer Temperatur (30 ° C oder höher). Notieren Sie die Anzahl der Nachkommen und ob jeder einzelne ist tot oder lebendig. Notieren Sie sich die rotifer als tot, wenn Zilien Bewegung der Korona vollständig zum Stillstand gekommen ist. Anmerkung: Rotifers befestigen häufig an den Seitenwänden der Vertiefungen. Vorsichtiges Pipettieren des Wassers hilft, sie zu finden. Wenn rotifers nicht gefunden oder are versehentlich durch Pipettieren beschädigt, sie aufzunehmen als &quot;zensiert&quot;, nicht als &quot;tot&quot;. </li><li> Entfernen Neugeborenen, wenn experimentelle rotifers aktiv reproduzieren sind. Neugeborenen wachsen schnell und es ist manchmal schwierig, sie aus experimentellen Rotatorien zu unterscheiden. </li></ol> 5. Datenanalyse <ol><li> Erstellen Sie die Kaplan-Meier - Überlebenskurve (Abbildungen 1 und 2) durch kumulative Überlebensrate auf der Y - Achse und die Zeit auf der X - Achse aufgetragen ist . Dies ist die häufigste Darstellung der Überlebensdaten. Verwenden Sie den nicht-parametrischer Log-Rank - Test auf statistische Vergleich der Überlebenszeit 34 (auch Mantel-Cox - Test genannt). Die Log-Rank-Test wird auch in anderen statistischen Standardpakete wie JMP und R. enthalten Hinweis: da Normalverteilung 35 durch das Überleben Daten erfüllt in der Regel nicht nicht Student-t-Test oder Varianzanalyse (ANOVA) , gefolgt von parametrischer mehrere Vergleich verwenden. Auch sind diese methods nehmen nicht zensiert Personen berücksichtigt. Mann-Whitney U-Test kann verwendet werden, wenn es keine zensierten Daten ist. </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.

Das aktuelle Protokoll beschreibt ein Verfahren zur in Brachionus Rotatorien die Überlebenszeit gemessen wird . Der entscheidende Schritt ist die Synchronisation von rotifer Bedingungen über mehrere Generationen. Bei experimentellen rotifers gut synchronisiert sind, ist eine typische Typ - I - Überlebenskurve mit sehr wenig frühe Sterblichkeit beobachtet , wie 18,24,37,38 in mehreren früheren Studien berichtet. Die Standardabweichungen der Überlebenszeit werden deshalb kleiner im Vergleich zu schlecht synchronisierten Rotatorien, in hoher statistischer Aussagekraft zur Folge hat. Die Synchronisation wird auch die Reproduzierbarkeit der Überlebenszeitmessungen zu erhöhen erwartet - weil Mütter unter optimalen Bedingungen kultiviert werden, die aktuelle Protokoll Offsets möglich schädlichen Auswirkungen der mütterlichen Generationen. Wenn frühe Sterblichkeit noch nach sorgfältiger Synchronisation beobachtet wird, sollten Sie neu vorbereitet Kulturmedien verwenden, eine andere Menge Fütterung Algen, oder eine neu gegründete experimentelle Kohorte (dhStart von Protokoll 3.1). Eine Einschränkung dieses Protokolls besteht darin, dass die gut synchronisiert Rotatorien sind potentiell überempfindlich. Zum Beispiel beim Screening von Chemikalien , die Lebensdauer verlängern, einige durch dieses Protokoll gescreent Chemikalien versagen kann erhebliche Auswirkungen auf die Lebensdauern von schlecht synchronisierten Rotatorien (zB Personen aus wilden und Batch-kultivierten Populationen) zu erkennen. Somit sollten die Ergebnisse dieser Versuche mit Vorsicht interpretiert werden. Die Wirkung des mütterlichen Alters auf die Nachkommen , die Überlebenszeit wurde auch in anderen wirbellosen Modelle einschließlich der Fruchtfliege Drosophila melanogaster und dem Nematoden Caenorhabditis elegans 39,40 berichtet. Obwohl es zeitraubend in dieser langlebigen Modelle ist, wäre die Synchronisationsprozedur über mehrere Generationen, die für diese Tiere in der Messung der Überlebenszeit experimentellen Schwankungen zu verringern.

Rotifers sind mikroskopisch kleine kosmopolitische Zooplankton (&lt;1 mm), die die phylum Rotatorier 1 darstellen. Sie haben eine einfache Körperplan von ca. 1.000 somatischen Zellen besteht, sowie eine charakteristische radähnlichen ciliary Vorrichtung die corona genannt, die für die Fortbewegung und Fütterung verwendet wird. Die meisten rotifers gehören zu den Klassen Monogononta oder Bdelloida, die etwa 1600 und 500 Arten enthalten jeweils 2. Monogononta rotifers haben in der Regel sowohl sexuelle und asexuelle Reproduktionsphasen (zyklische Parthenogenese), während Bdelloid Rotatorien durch obligatorische Parthenogenese 3 reproduzieren. Es ist somit möglich, genetisch identische rotifer Individuen zu erhalten, die in Experimenten eine hohe Reproduzierbarkeit gewährleistet. Darüber hinaus haben sie mehrere andere Vorteile als Modellorganismen, wie beispielsweise eine kurze Lebensdauer, Einfachheit der Kultur, die Verfügbarkeit von genomischen und transkriptomischen Sequenzdaten 4-7 und eine einzigartige phylogenetische Position entfernt von a8 rthropods und Nematoden. Rotifers versprechen daher invertebrate Modelle in ökologische, toxikologische und Alterungsuntersuchungen 12.09. Die Überlebenszeit unter der Exposition gegenüber Umweltstress oder Chemikalien ist ein häufig gemessene Parameter in diesen Forschungsfeldern 13-19. Allerdings ist Vorsicht geboten, wenn die Überlebenszeit von Rotatorien Messung, weil sie anfällig gegenüber Umweltbedingungen ihrer Mütter ist. Und zwar in der Monogononta rotifer Brachionus Manjavacas, weiblichen Nachkommen von im Alter von Mütter haben eine kürzere Lebensdauer als die von jungen Mütter; jedoch mütterliche Kalorienrestriktion (CR) kompensiert teilweise die schädlichen Auswirkungen von fortgeschrittenen Alter der Mutter 20. In B. plicatilis, mütterliche CR bietet Nachkommen Langlebigkeit, Beständigkeit lange Überlebenszeit unter Hunger und hohen oxidativen Stress verbunden mit erhöhter Expression von antioxidativen Enzymen 21,22. Das Alter der Mutter WirkungAuch in Bdelloid Rotatorien 23 beobachtet. Daher sollten die Bedingungen der experimentellen Rotatorien sorgfältig über mehrere Generationen vor der Messung der Überlebenszeit synchronisiert werden. Hier bieten wir ein Protokoll für die Messung der Überlebenszeit in Brachionus rotifers nach der Synchronisation von Kulturbedingungen über mehrere Generationen hinweg. Intermittierende Fasten (IF), eine Variation von CR wo rotifers periodisch zugeführt werden, um die Wirkung der Synchronisation aufgrund der bekannten Wirkung von IF auf Langlebigkeit 22,24 offenbaren angewendet.

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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.

Abbildung 1 zeigt repräsentative Überlebenskurven von schlecht synchronisierten Populationen (von zwei Wiederholungen). In diesem Experiment rotifers waren entweder gefüttert jeden Tag [ad libitum (AL) Gruppe] oder jeden zweiten Tag (IF - Gruppe). Das mediane Überleben betrug 13 und 18 Tage in der AL und IF-Gruppe. Obwohl es bekannt ist, dass, wenn die Lebensdauer der rotifer erstreckt, scheiterte dieser Versuch einen statistisch signifikanten Unterschied zwischen den Lebensdauern der AL zu erkennen und IF-Gruppen. Empirisch unzureichende Synchronisation Ergebnisse in der frühen Sterblichkeit und allmählichen Rückgang der Überlebensrate wie in diesem Experiment beobachtet. Schäden an Rotatorien durch unsachgemäße Behandlung oder niedrige Wasserqualität für die Subpopulation verursacht tendenziell ähnliche Ergebnisse zu erzielen. Wenn rotifer Bedingungen sind optimal und gut synchronisiert, ist frühe Sterblichkeit kaum beobachtet und accordingly Rotatorien neigen dazu , in einer synchronisierten Art und Weise während der späteren Phase des Experiments (Abbildung 2) sterben. Das mediane Überleben betrug 13 und 20 Tage in der AL und IF-Gruppe. Obwohl weniger Tiere als das Experiment in Figur 1 verwendet wurden, war der Unterschied in der Lebensdauer zwischen diesen Gruppen statistisch signifikant. Dies ist die repräsentative Ergebnisse aus mehr als fünf Experimenten , die vorher 22 veröffentlicht wurden. <img alt="Abbildung 1" src="/files/ftp_upload/54126/54126fig1.jpg" /> Abb . 1: Kaplan-Meier - Kurven für schlecht synchronisierte Personen zu intermittierenden Fastens (IF) unterworfen , um die AL - Gruppe wurde ad libitum während des gesamten Experiments eingespeist, während die ZF - Gruppe jeden zweiten Tag gefüttert wurde. N = 11 und N = 12 für das AL und IF-Gruppen jeweils (N bezieht sich auf die Anzahl der einzelnen in dem Experiment verwendet). Das Experiment wurde PERFORmed bei 25 ° C. Kein signifikanter Unterschied in der Lebensdauer festgestellt wurde , wenn Log-Rank - Test verwendet wurde (P = 0,1207). Allerdings ist diese Daten schwer zu interpretieren , da Log-Rank - Test nicht verwendet werden sollte , zwei sich kreuzende Überlebenskurven zu vergleichen , obwohl der Test bekannt ist robust 36 zu sein. Keine etablierten Methoden sind zur Zeit für die Überquerung Überlebenskurven mit zensierten Daten verfügbar. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/54126/54126fig2.jpg" /> Abbildung 2:. Kaplan-Meier - Kurven für synchronisierte Individuen ausgesetzt IF Die rotifer Kohorte, erhalten durch Vorkultur wurde der gleichen IF Zeitplan unterworfen (jeden zweiten Tag gefüttert). N = 6 und N = 8 für AL und IF-Gruppe. Der Versuch wurde bei 25 ° C durchgeführt wird. Log-Rank - Test, p = 0,0057. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54126/54126fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a>

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.

Die Messung der Überlebensdauer in<em&gt; Brachionus</em&gt; Rotifers: Synchronisation von Maternal Bedingungen

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

Nanyang Technological University

Research

JoVE Journal

Biology

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

8.4K Aufrufe.  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.

Serie: 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.

Zeitraffer-Mikroskopie der frühen Embryogenese 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 ...

Forschung

Biologie

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.

Grundlegende<em&gt; Caenorhabditis elegans</em&gt; Methoden: Synchronisation und 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 ...

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.

Formulierungen für die Gefriertrocknung von Bakterien und deren Einfluss auf Zellebensdauer

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.

Quantitative Messung der Invadopodia-vermittelte extrazellulären Matrix Proteolyse in Einzel-und Multizelluläre Contexts

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.

Messung der Lebensdauer 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.

Die Messung der Hämsynthese Levels in Säugerzellen

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.

Messung der Stoffwechselrate in<em&gt; Drosophila</em&gt; Mit Respirationstest

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.

Sonde-basierte Echtzeit-PCR Ansätze zur quantitativen Messung von 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.

Synchronisation<em&gt; Caulobacter crescentus</em&gt; Für die Untersuchung der Bakterienzellzyklus

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.

Zeitliche Verfolgung von Zellzyklus mittels Durchflusszytometrie, ohne die Notwendigkeit für die Synchronisation

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