Ce travail a été soutenu par des subventions de la NIA à YW.CF (AG21068, AG31086).

1. Préparation de la solution d&#39;insuline <ol><li> Préparer une nouvelle solution insuline bovine en dissolvant l&#39;insuline dans le PBS pour obtenir la concentration de 0,01 mg / ml. L&#39;insuline / PBS et le contrôle des solutions PBS doit être conservé sur la glace pendant toute la procédure d&#39;injection. Ces solutions doivent être préparées avec 0,5% (v / v) bleu FD &amp; C non. 1 colorant alimentaire. </li></ol> 2. Préparation d&#39;aiguille et l&#39;injection Set-up <ol><li> Préparer des aiguilles de verre capillaire en utilisant une micropipette puller.The réglages suivants extracteur produire des aiguilles d&#39;une qualité suffisante pour l&#39;injection: la chaleur, 345; Pull, 210; vitesse, 100; Temps, 200 (100ms). Aiguilles fraîchement tiré doit être émoussé en appuyant sur la pointe grâce à un tissu Kimwipe. Ce processus supprime émousser la pointe allongée de résistance élevée et produit une pointe plus robuste avec un diamètre plus grand pore. </li><li> Placez les aiguilles émoussées tip-up dans un microtube contenant de l&#39;insuline / solution de PBS ou du PBS seul. Capillaire résultats de l&#39;action en arrière-remplissage de chaque aiguille. Observez la pointe de l&#39;aiguille sous un stéréomicroscope pour s&#39;assurer qu&#39;aucune bulle d&#39;air des débris ou apparaître au bout. Jeter les aiguilles qui ne remplissent pas proprement. </li><li> Insérer des aiguilles remplies dans le porte-aiguille microinjecteur manuel et la position du porte-aiguille avec un micromanipulateur sorte que l&#39;aiguille est visible à travers une loupe binoculaire. Appliquer une pression positive sur la colonne de liquide dans l&#39;aiguille en tournant le bouton du micromètre manuel microinjecteur attaché au piston de la seringue microinjecteur. </li><li> Vérifiez que la pression suffisante pour le déplacement du liquide a été appliquée en touchant un Kimwipe à la pointe de l&#39;aiguille et la confirmation de l&#39;écoulement du fluide. </li><li> Une fois que l&#39;aiguille a été préparé pour l&#39;injection, apposer un tableau d&#39;étalonnage de la tige de l&#39;aiguille avec du ruban adhésif clair, et amener la pointe de l&#39;aiguille en lumière à travers une loupe binoculaire sous un grossissement de 20x. </li></ol> 3. Préparation Fly <ol><li> Dix jours d&#39;âge mouches femelles sont utilisées pour des expériences d&#39;injection. Recueillir des mouches dans les 24 h de l&#39;éclosion. Anesthetize mouches avec du CO 2 humidifié sur un coussin de gaz, trier les mâles et les mouches femelles lieu dans des fioles contenant un étalon ou d&#39;un régime de traitement. Maintenir mouches femelles sur les régimes expérimentaux pendant 10 jours. </li><li> Sur le dixième jour suivant l&#39;éclosion et la séparation sur les régimes alimentaires expérimentaux, le transfert des mouches de leurs produits alimentaires contenant des flacons dans des flacons contenant 5 ml d&#39;une fiche d&#39;agar 2% et les affamer pour les 12-16 h. </li><li> Transfert des mouches affamées dans des flacons contenant des filtres de glucose à 10% à tremper pendant 1 heure avant l&#39;injection d&#39;insuline. </li><li> Brièvement anesthésier les mouches avec du CO 2 humidifié après le repas du glucose puis d&#39;immobiliser froide eux sur la glace. </li></ol> 4. Procédure d&#39;injection <ol><li> Saisir délicatement la mouche à froid immobilisé avec une paire de pinces fines et maintenez-le à proximité de la pointe de l&#39;aiguille pour que la pointe est adjacente à la région antérieure du côté gauche du thorax de la mouche. Réunir les deux bout de l&#39;aiguille et le thorax de la mouche en lumière sous la loupe binoculaire. </li><li> Déplacez doucement la volée vers la pointe de l&#39;aiguille de sorte que l&#39;aiguille touche le centre de la protubérance de la région du thorax prescutum laissé la mouche (Fig. 1). Une fois que la mouche est correctement orientés et alignés avec la pointe de l&#39;aiguille, continuer à faire avancer la volée sur l&#39;aiguille pour que l&#39;aiguille empale le centre de la prescutum. </li><li> Appliquer une pression positive, au besoin en faisant progresser le piston de la seringue avec le bouton micromanipulateur l&#39;microinjecteur jusqu&#39;au 0,1 l de liquide a été injecté dans la volée. L&#39;écoulement de fluide dans les hemocoel la mouche va donner une couleur bleue sur le côté gauche du thorax antérieur. Parfois on doit se rétracter et l&#39;avance à plusieurs reprises pour déloger toute obstruction pointe de l&#39;aiguille et de permettre l&#39;écoulement du fluide. </li><li> Autoriser mouches injecté à récupérer dans des flacons de gélose à 2% pour les points de temps désigné avant l&#39;extraction hémolymphe. </li></ol> 5. Collection hémolymphe <ol><li> Brièvement anesthésier les mouches avec du CO 2 à humidifié désiré points de temps après l&#39;injection et la position de chaque mouche dorso-latéral vers le bas sur un ruban double face apposée sur la surface d&#39;un CO 2 pad. Organiser des mouches, même dans les lignes avec des capsules tête alignée. On peut utiliser un bâton applicateur garni de bois pour faire pression sur leurs ailes dans le ruban adhésif et assurer l&#39;immobilisation. </li><li> Une fois que les mouches sont fixés à la bande, saisir proboscis chaque mouche avec une pince fine et tirez vers la région ventrale et postérieure de la mouche de telle sorte que l&#39;avant de la capsule de la tête est visible à travers le microscope stéréo. </li><li> Avec votre autre main et tout en maintenant la trompe en position, percer le centre de la capsule de la tête juste au-dessus de la suture ptilinal aide d&#39;une aiguille de tungstène pointu (Fig. 2). Il faut être prudent à ne pas insérer l&#39;aiguille trop loin car il est facile de frapper la pointe de l&#39;aiguille à travers l&#39;autre côté de la capsule céphalique et perdre contrôle des flux de l&#39;hémolymphe. Crevaison mouches le tout dans un groupe d&#39;injection avant de recueillir l&#39;hémolymphe. </li><li> Une fois la capsule céphalique a été perforé, utilisez un bâton applicateur garni de bois pour appliquer doucement la pression à l&#39;abdomen de la mouche. Il peut être utile de rouler les survoler afin qu&#39;il repose sur sa face ventrale que vous appliquez une pression. Cette procédure va produire une goutte d&#39;hémolymphe du site de ponction tête de la capsule. </li><li> Toucher une extrémité d&#39;un tube de 1 microlitre microcapillaire à la goutte d&#39;hémolymphe émergents du site de ponction tête de la capsule. L&#39;hémolymphe va entrer dans le tube par capillarité. Déterminer et noter la quantité d&#39;hémolymphe recueillies en cochant la colonne de fluide dans le tube microcapillaire contre un tableau de volume graduée. </li></ol> 6. Détermination du glucose hémolymphe <ol><li> Ejecter échantillons hémolymphe en microplaque contenant 100 ul de réactif glucose Infinity. Gardez la plaque sur la glace pendant le chargement des échantillons d&#39;hémolymphe. </li><li> Établir une courbe standard en chargeant séparément 1 pl chacune des solutions mères de glucose à 50 mM, 25mm, 12.5mm, 6.25mm et 3.125mM. </li><li> Incuber les échantillons à 37 ° C pendant 10 minutes. </li><li> Détecter l&#39;absorbance à 340 nm. </li><li> Compte pour le volume de l&#39;hémolymphe recueillies et déterminer la concentration en glucose de l&#39;échantillon basée sur la courbe standard avec des concentrations de glucose connue. </li></ol> 7. Les résultats représentatifs Une réponse à l&#39;insuline typiques de tolérance est détecté dans l&#39;insuline injectée-mouches, où une baisse des taux circulants de glucose est détecté 15 minutes après l&#39;injection. En revanche, une telle réponse n&#39;est pas vu dans mouches PBS injecté (Fig. 3). Cette réponse de l&#39;élimination du glucose périphériques continue de l&#39;insuline injectée-mouches jusqu&#39;à 30 minutes après l&#39;injection. Nous faisons régulièrement l&#39;extrait de 0,2 à 0,5 l de l&#39;hémolymphe par 4-5 mouches dans chaque groupe d&#39;injection. Trois groupes d&#39;injection sont inclus dans chaque expérience. <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="Figure 1"/> Figure 1. Le côté gauche du thorax montrant la drosophile site d&#39;insertion d&#39;aiguille (modification du Demerec, 1950) 7. Insérez l&#39;aiguille dans le centre de la prescutum sur la partie antérieure, région dorsale du côté gauche du thorax. <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="Figure 2"/> Figure 2. Vue frontale de la tête de la drosophile, montrant l&#39;emplacement de ponction pour l&#39;extraction de l&#39;hémolymphe (modification du Demerec, 1950) 7. Piquer la capsule de la tête avec une sonde de tungstène finement aiguisé dans le centre de la capsule de la tête juste au-dessus de la suture ptilinal. <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="Figure 3"/> Figure 3. Une réponse à l&#39;insuline typiques de tolérance détecté chez les adultes contre les mouches. Contrôle des mouches w 1118 ont été injectés avec de l&#39;insuline bovine (1 ng dans PBS) ou PBS. Les mouches dans des groupes de reproduire ont ensuite été autorisés à récupérer pour les minutes 0, 15 ou 30 et les niveaux de glucose circulant ont été mesurés.

<ol>
	<li>Rulifson, E. J., Kim, S. K., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+insulin-producing+neurons+in+flies:+growth+and+diabetic+phenotypes.">Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes.</a> Science. 296, 1118-1118 (2002).</li><li>Kim, S. K., Rulifson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+mechanisms+of+glucose+sensing+and+regulation+by+Drosophila+corpora+cardiaca+cells.">Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells.</a> Nature. 431, 316-316 (2004).</li><li>Broughton, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longer+lifespan,+altered+metabolism,+and+stress+resistance+in+Drosophila+from+ablation+of+cells+making+insulin-like+ligands.">Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.</a> Proc Natl Acad Sci U S A. 102, 3105-3105 (2005).</li><li>Haselton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+ablation+of+adult+Drosophila+insulin-producing+neurons+modulates+glucose+homeostasis+and+extends+life+span+without+insulin+resistance.">Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance.</a> Cell Cycle. 9, 3063-3063 (2010).</li><li>Haselton, A. T., Fridell, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Drosophila+melanogaster+as+a+model+for+the+study+of+glucose+homeostasis.">Adult Drosophila melanogaster as a model for the study of glucose homeostasis.</a> Aging (Albany NY). 2, 523-523 (2010).</li><li>Belgacem, Y. H., Martin, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+insulin+pathways+alters+trehalose+level+and+abolishes+sexual+dimorphism+in+locomotor+activity+in+Drosophila.">Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila.</a> J Neurobiol. 66, 19-19 (2006).</li><li>Demerec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Drosophila. , (1950).</li></ol>

La technique décrite dans ce rapport est potentiellement utile dans toute étude qui examine les processus physiologiques entraînant des altérations décelables dans la composition de l&#39;hémolymphe de drosophile. En combinant l&#39;injection et la collecte de l&#39;hémolymphe de cette manière, il est possible de déterminer les effets immédiats physiologiquement pertinents d&#39;un traitement expérimental particulier ou de manipulation. Le principal avantage de cette «saignée» la technique dans la...

<table border="1"><tbody><tr><th> Nom du réactif / équipements </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> L&#39;insuline bovine </td><td> Sigma </td><td> I5500 </td></tr><tr><td> Réactif du glucose Infinity </td><td> Thermo Electron Corporation </td><td> TR1541 </td></tr><tr><td> Manuel microinjecteur </td><td> Instrument Sutter </td><td></td></tr><tr><td> P-87 Flamming / Brown micropipette extracteur </td><td> Instrument Sutter </td><td></td></tr><tr><td> Simple cylindre en verre de borosilicate capillaires </td><td> Systèmes AM </td><td> 626000 </td></tr><tr><td> FD &amp; C bleu n º 1 </td><td> McCormick &amp; Company </td><td></td></tr><tr><td> 1 pl tubes microcapillaires </td><td> Drummond </td><td></td></tr><tr><td> Micromanipulateur manuelle à trois axes et de la base </td><td> Instruments de précision du monde </td><td></td></tr></tbody></table>

insulin injection and hemolymph extraction to measure insulin sensitivity in adult drosophila melanogaster

Conservé en nutriments des mécanismes de détection existent entre les mammifères et la mouche des fruits, où les peptides ressemblant d&#39;insuline et de glucagon chez les mammifères, respectivement la fonction de maintenir l&#39;homéostasie du glucose au cours du développement 1,2 stades larvaires. Les études sur les mouches adultes largement post-mitotiques ont révélé une perturbation de l&#39;homéostasie du glucose comme le résultat d&#39;ablation génétique de l&#39;insuline-like peptide (PAI) de cellules productrices (IPC) 3. Ainsi, les mouches à fruits pour adultes sont très prometteurs en tant que système modèle génétique adapté à des troubles métaboliques, y compris le diabète de type II. Pour développer davantage le système de la mouche des fruits, des dosages physiologiques comparables utilisés pour mesurer la tolérance au glucose et la sensibilité à l&#39;insuline chez les mammifères doivent être établis. À cette fin, nous avons récemment décrit une nouvelle procédure pour la mesure de la réponse orale de tolérance au glucose chez la mouche adulte et démontré l&#39;importance d&#39;IPC pour adultes dans le maintien de l&#39;homéostasie du glucose 4,5. Ici, nous avons modifié la procédure décrite précédemment pour 6 injections d&#39;insuline et l&#39;a combiné avec une méthode d&#39;extraction hémolymphe roman pour mesurer la sensibilité périphérique à l&#39;insuline chez la mouche adulte. Unique, notre protocole permet des mesures physiologiques directs de la capacité de la mouche adulte est de disposer d&#39;une charge en glucose périphériques après injection d&#39;insuline, une méthodologie qui rend possible de caractériser les mutants signalisation de l&#39;insuline et les interventions possibles concernant la tolérance au glucose et la sensibilité à l&#39;insuline chez la mouche adulte.

Watch this Scientific Journal Video about Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster at JoVE.com

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Signalisation de l&#39;insuline conservées voies trouvés dans la mouche des fruits<em&gt; Drosophila melanogaster</em&gt; Faire de cet organisme un outil potentiel pour la modélisation des troubles métaboliques, notamment le diabète de type II. À cette fin, il est essentiel d&#39;établir des analyses physiologiques pour mesurer efficacement l&#39;insuline d&#39;action systémique dans l&#39;élimination du glucose périphérique dans la mouche adulte.

Injection d&#39;insuline et d&#39;extraction hémolymphe pour mesurer la sensibilité d&#39;insuline dans les adultes Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

insulin-injection-hemolymph-extraction-to-measure-insulin-sensitivity

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Biology

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

19.6K vues.  State University of New York. Signalisation de l'insuline conservées voies trouvés dans la mouche des fruits Drosophila melanogaster Faire de cet organisme un outil potentiel pour la modélisation des troubles métaboliques, notamment le diabète de type II. À cette fin, il est essentiel d'établir des analyses physiologiques pour mesurer efficacement l'insuline d'action systémique dans l'élimination du glucose périphérique dans la mouche adulte.

Vidéo: Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Suivi vidéo haute résolution de la locomotion chez les adultes Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

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Biologie

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissection de Oenocytes d&#39;adultes Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Un dosage quantitatif de l&#39;insuline exprimer formant colonie Progéniteurs

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

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.

Mesure de la durée de vie en<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 ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

L&#39;utilisation du test Ramsay mesurer la sécrétion de liquide et de flux d&#39;ions dans le Tarifs<em&gt; Drosophila melanogaster</em&gt; Tubule Malpighian

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Mesure de l&#39;absorption de glucose et réponse à la stimulation de l&#39;insuline dans<em&gt; In Vitro</em&gt; Myotubes primaires humains cultivés

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

Confocale, imagerie du Neuropeptide Y-pHluorin : une Technique pour visualiser l’insuline granules cytotoxiques dans les îlots murins et humains intacts

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Détermination de zone optique transversale musculaire des Muscles de vol Indirect adulte de Drosophila Melanogaster

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Oral Infection bactérienne et excrétion chez Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualisation et suivi des ARNm endogènes dans Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...