Diese Arbeit wurde durch Zuschüsse aus dem NIA zu YW.CF (AG21068, AG31086) unterstützt.

1. Insulin Herstellung der Lösung <ol><li> Bereiten Sie frische Rinder-Insulin durch Auflösen von Insulin in PBS, um die Konzentration von 0,01 mg / ml zu erreichen. Sowohl Insulin / PBS und Kontrolle PBS-Lösungen müssen auf dem Eis während der Injektion gehalten werden. Diese Lösungen sollten mit 0,5% (v / v) FD &amp; C Blau nicht vorbereitet werden. 1 Nahrung Färbung. </li></ol> 2. Needle Vorbereitung und Injection Set-up <ol><li> Bereiten Kapillare Glasnadeln mit einer Mikropipette puller.The folgenden puller Einstellungen erzeugen Nadeln von ausreichender Qualität für die Injektion: Hitze, 345; Pull, 210; Velocity, 100; Zeit, 200 (100ms). Frisch gezapftes Nadeln müssen durch Drücken der Spitze durch eine Kimwipe Gewebe abgestumpft werden. Dieses Abstumpfen-Verfahren beseitigt die längliche hohe Spitze und erzeugt ein dicker Spitze mit einem größeren Porendurchmesser. </li><li> Legen Sie abgestumpft Nadeln tip-up in ein Mikrozentrifugenröhrchen mit dem Insulin / PBS-Lösung oder PBS allein. Kapillarwirkung Ergebnisse in Back-Füllung jeder Nadel. Beachten Sie die Spitze der Nadel unter einem Stereomikroskop, um sicherzustellen, dass keine Fremdkörper oder Luftbläschen an der Spitze erscheinen. Entsorgen Sie alle Nadeln, die nicht ganz gefüllt ist sauber. </li><li> Legen gefüllt Nadeln in die manuelle Mikroinjektor Nadelhalter und die Position der Nadelhalter mit einem Mikromanipulator, so dass die Nadelspitze sichtbar durch ein Stereomikroskop. Bewerben positiven Druck auf die Flüssigkeitssäule in der Nadel durch Drehen des manuellen Mikroinjektor Mikrometer-Regler an den Kolben auf die Mikroinjektor Spritze. </li><li> Überprüfen Sie, ob genügend Druck für Fluid-Verschiebung ist durch Berühren eines Kimwipe an der Spitze der Nadel und der Bestätigung Strömung angewendet worden. </li><li> Sobald die Nadel für die Injektion vorbereitet, ein Messprotokoll anzubringen, um die Nadel Welle mit durchsichtigem Klebeband und bringen Sie die Nadelspitze in den Fokus durch ein Stereomikroskop unter 20-facher Vergrößerung. </li></ol> 3. Fly Vorbereitung <ol><li> Zehn Tage alte weibliche Fliegen sind für die Injektion Experimente verwendet. Sammeln Sie fliegt innerhalb von 24 h Schlüpfen. Anesthetize fliegt mit befeuchteten CO 2 auf einem Gas-Pad, aussortieren Männchen und stellen die weiblichen Fliegen in Fläschchen mit Standard-oder die Behandlung Ernährung. Behalten weibliche Fliegen auf experimentellen Diäten für 10 Tage. </li><li> Am zehnten Tag nach Schlüpfen und die Trennung auf Versuchsdiäten, Fliegen übertragen aus ihrer Nahrung-haltige Fläschchen zu Fläschchen mit einem 5 ml-Stecker mit 2% Agar und verhungern sie für 12-16 h. </li><li> Transfer verhungert fliegt Ampullen mit 10% Glucose getränkten Filter für 1 Stunde vor Insulininjektion. </li><li> Kurz betäuben die Fliegen mit befeuchteten CO 2 nach der Glucose-Zufuhr und dann kalt immobilisieren sie auf Eis. </li></ol> 4. Injektionsverfahren <ol><li> Fassen Sie ein kalt-immobilisierten fliegen mit einer feinen Pinzette und halten Sie die Nadelspitze in der Nähe, so dass die Spitze befindet sich neben dem vorderen Bereich der linken Seite der Fliege Thorax. Bringen Sie beide die Nadelspitze und die Fliege Thorax in den Fokus unter dem Stereomikroskop. </li><li> Bewegen Sie die fliegen in Richtung der Nadelspitze, so dass die Nadelspitze berührt das Zentrum der Höcker des linken prescutum Region der Fliege Thorax (Abb. 1). Nachdem die Fliege korrekt ausgerichtet ist und im Einklang mit der Nadelspitze, weiterhin die fliegen auf die Nadel so, dass die Nadel die Mitte des prescutum spießt Voraus. </li><li> Bewerben positive Druck durch Vorschieben des Spritzenkolbens mit der Mikroinjektor der Mikromanipulator Knopf nötig, bis 0,1 ul der Flüssigkeit in-the-fly injiziert wurde. Die Strömung der Flüssigkeit in der Fliege hemocoel wird eine blaue Farbe auf die linke Seite des vorderen Thorax zu vermitteln. Gelegentlich muss man einfahren und vorab mehrmals, um alle Nadelspitze Blockade lösen und ermöglichen eine Strömung. </li><li> Lassen Sie injizierten fliegt in 2% Agar Fläschchen für bestimmte Zeitpunkte vor Hämolymphe Extraktion erholen. </li></ol> 5. Hämolymphe Sammlung <ol><li> Kurz betäuben fliegt mit befeuchteten CO 2 in der gewünschten Nacheinspritzung Zeitpunkten und Position jedes fliegen dorsal-side-down auf doppelseitigem Klebeband angebracht, um die Oberfläche eines CO 2-Pad. Vereinbaren Sie fliegt in geraden Reihen mit Kopf Kapseln ausgerichtet. Man kann mit einem getrimmt Holzstab stick, ihre Flügel in die Band Kleber drücken und sicherstellen, Immobilisierung. </li><li> Sobald Fliegen sind, um das Band fixiert, fassen jeweils Fliege Rüssel mit feinen Pinzetten und ziehen Sie es vorsichtig in Richtung der ventralen und dann hinteren Bereich der Fliege, so dass die Vorderseite des Kopfes Kapsel ist sichtbar durch die Stereo-Mikroskop. </li><li> Mit der anderen Hand, und halten den Rüssel in Position, Punktion der Mitte des Kopfes Kapsel oberhalb der ptilinal Naht mit einem scharfen Wolfram-Nadel (Abb. 2). Man muss vorsichtig sein, die Kanüle nicht zu weit einfügen, wie es leicht ist, die Spitze der Nadel durch die andere Seite der Kopfkapsel Punsch und später verlieren controlle der Hämolymphe fließen. Punktion alle Fliegen in eine Spritze Gruppe vor dem Sammeln Hämolymphe. </li><li> Sobald der Kopf Kapsel durchstochen hat, verwenden Sie eine abgespeckte Holzstab Stick drücken Sie leicht auf der Fliege Bauch. Es kann hilfreich sein, die Fliege um, so dass es beruht auf der Bauchseite, wie Sie Druck ausüben zu rollen. Dieses Verfahren wird ein Tröpfchen Hämolymphe aus der Kopfkapsel Einstichstelle zu produzieren. </li><li> Berühren Sie ein Ende des 1μl Mikrokapillare Rohr auf die Hämolymphe Tropfen, die aus der Kopfkapsel Einstichstelle. Die Hämolymphe wird das Rohr durch Kapillarwirkung geben. Ermitteln und notieren Sie die Menge der Hämolymphe, indem Sie die Flüssigkeitssäule im Mikrokapillare Rohr gegen ein abgestuftes Volumen-Chart gesammelt. </li></ol> 6. Hämolymphe Glucose Bestimmung <ol><li> Eject Hämolymphe Proben in Mikrotiterplatten Vertiefungen mit 100 ul von Infinity Glucosereagenz. Halten Platte auf Eis beim Laden Hämolymphe Proben. </li><li> Stellen Sie eine Standard-Kurve durch das Laden separat 1 ul jeder von Glukose Stammlösungen bei 50mm, 25mm, 12,5 mm, 6,25 mm und 3.125mM. </li><li> Inkubieren Proben bei 37 ° C für 10 Minuten. </li><li> Detect Absorption bei 340 nm. </li><li> Konto für Hämolymphe Volumen gesammelt und bestimmen Probe Glucose-Konzentration auf die Standardkurve mit bekannten Glukosekonzentrationen basiert. </li></ol> 7. Repräsentative Ergebnisse Ein typisches Insulintoleranz Antwort ist in der Insulin-Injektion fliegt, wo ein Rückgang der zirkulierenden Glukosespiegel 15 Minuten nach der Injektion erfasst wird. Im Gegensatz dazu ist eine solche Reaktion nicht in PBS injiziert Fliegen (Abb. 3). Diese Reaktion im peripheren Glukose zur Verfügung weiterhin in der Insulin-Injektion fliegt bis zu 30 Minuten nach der Injektion. Wir routinemäßig Extrakt 0,2-0,5 ul Hämolymphe pro 4-5 Fliegen in jeder Injektion Gruppe. Drei Injektion Gruppen sind in jedem Experiment einbezogen. <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Linke Seite des Drosophila Thorax zeigt Nadel Insertionsstelle (Modified aus Demerec, 1950) 7. Legen Sie die Nadel durch die Mitte des prescutum auf der vorderen, hinteren Bereich der linken Seite des Thorax. <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Frontansicht des Drosophila Kopf zeigt Punktionsstelle für Hämolymphe Extraktion (modifiziert nach Demerec, 1950) 7. Punktion der Kopfkapsel mit einer fein geschliffen Wolfram-Sonde in die Mitte des Kopfes Kapsel oberhalb der ptilinal Naht. <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="Abbildung 3"/> Abbildung 3. Ein typisches Insulintoleranz Reaktion unter Kontrolle Erwachsenen erkannt Fliegen. Control-w 1118 Fliegen wurden mit Rinder-Insulin (1 ng in PBS) oder PBS nur injiziert. Fliegen in replizieren Gruppen wurden dann für 0, 15 oder 30 Minuten und zirkulierenden Glukosespiegel wieder gemessen.

<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>

Die Technik in diesem Bericht beschrieben wird, potentiell nützlich in jeder Studie, die physiologischen Prozesse sich in nachweisbaren Veränderungen in Drosophila Hämolymphe Zusammensetzung untersucht. Durch die Kombination von Injektions-und Hämolymphe Sammlung auf diese Weise ist es möglich, die unmittelbare physiologisch relevanten Auswirkungen einer bestimmten experimentellen Behandlung oder Manipulation zu ermitteln. Der primäre Vorteil dieser &quot;Aderlass&quot;-Technik in Hämolymphe Sammlung im ...

<table border="1"><tbody><tr><th> Name des Reagenzes / Ausrüstung </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> Rinderinsulin </td><td> Sigma </td><td> I5500 </td></tr><tr><td> Infinity Glukose-Reagenz </td><td> Thermo Electron Corporation </td><td> TR1541 </td></tr><tr><td> Manuelle Mikroinjektor </td><td> Sutter Instrument </td><td></td></tr><tr><td> P-87 Flamming / Brown Mikropipette puller </td><td> Sutter Instrument </td><td></td></tr><tr><td> Single Barrel Borosilikat Kapillare aus Glas </td><td> AM-Systeme </td><td> 626000 </td></tr><tr><td> FD &amp; C Blau Nr. 1 </td><td> McCormick &amp; Company </td><td></td></tr><tr><td> 1 ul Mikrokapillare Rohre </td><td> Drummond </td><td></td></tr><tr><td> Drei-Achs-Handbuch Mikromanipulator und Base </td><td> World Precision Instruments </td><td></td></tr></tbody></table>

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

Konservierte Nährstoff sensing Mechanismen existieren zwischen Säugetier und Fruchtfliege, wo Peptide ähnlich Säugetieren Insulin und Glukagon bzw. Funktion, um Glukose-Homöostase während der Entwicklung Larvenstadien 1,2 zu halten. Studien zu weitgehend post-mitotischen erwachsenen Fliegen haben Störung des Glukose-Homöostase als Ergebnis des genetischen Ablation von Insulin-like peptide (ILP) produzierenden Zellen (IPCs) 3 enthüllt. So halten erwachsenen Fruchtfliegen großes Versprechen als eine geeignete genetische Modellsystem für Stoffwechselstörungen wie Diabetes Typ II. Zur weiteren Entwicklung der Fruchtfliege System, müssen vergleichbare physiologische Tests verwendet werden, um die Glukosetoleranz und Insulinsensitivität bei Säugetieren messen eingerichtet werden. Zu diesem Zweck haben wir kürzlich ein neues Verfahren zur Messung der oralen Glukosetoleranztest Reaktion in die erwachsene Fliege beschrieben und demonstriert die Bedeutung der Erwachsenenbildung IPCs bei der Aufrechterhaltung Glukosehomöostase 4,5. Hier haben wir ein zuvor beschriebenes Verfahren zur Insulin-Injektion 6 modifiziert und kombiniert es mit einem neuartigen Hämolymphe-Extraktions-Verfahren der peripheren Insulinsensitivität in der erwachsenen Fliegen zu messen. Einzigartig ist, ermöglicht es unseren Protokoll direkte physiologische Messungen der Erwachsenen fliegen die Fähigkeit eines peripheren Glukose-Last auf Insulin-Injektion, eine Methodik, die es möglich, Insulin-Signalkaskade Mutanten und möglichen Interventionen beeinflussen Glukosetoleranz und Insulinsensitivität in die erwachsene Fliege zu charakterisieren, macht entsorgen.

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

Konservierte Insulin-Signalwege in der Fruchtfliege gefunden<em&gt; Drosophila melanogaster</em&gt; Make dieser Organismus ein potentielles Werkzeug für die Modellierung von Stoffwechselerkrankungen wie Typ II Diabetes. Zu diesem Zweck ist es wichtig, physiologische Tests zu etablieren, um effektiv zu messen systemischen Wirkung von Insulin in peripheren Glukose zur Verfügung in die erwachsene Fliege.

Insulin-Injektion und Hämolymphe Extraction die Insulinsensitivität in Adult Measure 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

Research

JoVE Journal

Biology

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

19.6K Aufrufe.  State University of New York. Konservierte Insulin-Signalwege in der Fruchtfliege gefunden Drosophila melanogaster Make dieser Organismus ein potentielles Werkzeug für die Modellierung von Stoffwechselerkrankungen wie Typ II Diabetes. Zu diesem Zweck ist es wichtig, physiologische Tests zu etablieren, um effektiv zu messen systemischen Wirkung von Insulin in peripheren Glukose zur Verfügung in die erwachsene Fliege.

Serie: 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.

Hochauflösendes Video Tracking von 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 ...

Forschung

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.

Präparation Oenocytes von Adult 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.

A Quantitative Assay für Insulin-exprimierenden Koloniebildende Vorläufern

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.

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 ...

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.

Die Nutzung der Ramsay Assay zu Flüssigkeitssekretion und Ionenfluss Preise in der Maßnahme<em&gt; Drosophila melanogaster</em&gt; Malpighischen Tubulus

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.

Glukoseaufnahme Messung und Response auf Insulin Stimulation in<em&gt; In vitro</em&gt; Kultivierte menschliche primäre Myotubes

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.

Confocal Imaging von Neuropeptid Y-pHluorin: eine Technik, um Insulin Granulat Exozytose in intakten murinen und menschliche Inseln visualisieren

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.

Optische Cross-sectional Muskel Bereich Bestimmung von Drosophila Melanogaster Erwachsenen indirekten Flugmuskeln

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.

Mündliche bakterielle Infektion und 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 ...

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.

Visualisierung und Verfolgung endogenes mRNAs in Live Drosophila melanogaster Eierkammern

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