Questo lavoro è stato sostenuto dalle concessioni dal NIA a YW.CF (AG21068, AG31086).

1. L&#39;insulina Soluzione Preparazione <ol><li> Preparare una soluzione fresca bovina insulina sciogliendo insulina in PBS per raggiungere la concentrazione di 0,01 mg / ml. Sia insulina / PBS e controllo PBS soluzioni devono essere tenuti in ghiaccio per tutta la procedura di iniezione. Queste soluzioni devono essere preparate con il 0,5% (v / v) FD &amp; C non blu. 1 colorante alimentare. </li></ol> 2. Ago Preparazione e iniezione Set-up <ol><li> Preparare gli aghi capillare di vetro con un puller.The micropipetta seguenti impostazioni di estrattore produrre aghi di qualità sufficiente per l&#39;iniezione: Calore, 345; Pull, 210; Velocità, 100; Tempo, 200 (100 ms). Aghi appena tirato devono essere smussati premendo la punta attraverso un tessuto Kimwipe. Questo processo di ottundimento rimuove la punta allungata alta resistenza e produce una punta più robusto con un diametro dei pori più grandi. </li><li> Luogo aghi smussati punta-up in una provetta contenente l&#39;insulina / PBS soluzione o PBS solo. Capillare azione risulta in back-riempimento di ogni ago. Osservare la punta di un ago sotto uno stereomicroscopio per garantire che non detriti o bolle d&#39;aria comparire sulla punta. Eliminare qualsiasi aghi che non riempire correttamente. </li><li> Inserire aghi riempito nel manuale porta aghi microinjector e la posizione del titolare ago con un micromanipolatore in modo che la punta dell&#39;ago è visibile attraverso uno stereomicroscopio. Applicare una pressione positiva sul fluido nella colonna l&#39;ago ruotando la manopola manuale micrometrica microinjector attaccato al pistone sulla siringa microinjector. </li><li> Verificare che la pressione sufficiente per lo spostamento dei fluidi è stata applicata toccando un Kimwipe alla punta dell&#39;ago e confermando il flusso del fluido. </li><li> Una volta che l&#39;ago è stato preparato per l&#39;iniezione, apporre una tabella di calibrazione per l&#39;albero ago con del nastro adesivo trasparente, e portare la punta dell&#39;ago a fuoco attraverso uno stereomicroscopio con ingrandimento 20X. </li></ol> 3. Preparazione volare <ol><li> Le mosche femmina di dieci giorni di età sono usati per gli esperimenti di iniezione. Raccogliere le mosche entro 24 ore eclosion. Anestetizzare vola con umidificata CO 2 su un cuscinetto a gas, risolvere i maschi e vola posto femminile in fiale contenenti standard o una dieta di trattamento. Mantenere la femmina vola sulle diete sperimentale per 10 giorni. </li><li> Il decimo giorno successivo eclosion e separazione a diete sperimentali, il trasferimento di aria dal loro cibo contenenti fiale di fiale contenenti da 5 ml tappo del 2% agar e farli morire di fame per 12-16 h. </li><li> Trasferimento vola di fame fiale contenenti filtri di glucosio al 10% messi a bagno per 1 ora prima dell&#39;iniezione di insulina. </li><li> Brevemente anestetizzare le mosche con 2 CO umidificata dopo l&#39;alimentazione del glucosio e poi immobilizzare freddo li sul ghiaccio. </li></ol> 4. Procedura di iniezione <ol><li> Afferrare una mosca freddo immobilizzato con un paio di pinze sottili e tenerlo vicino alla punta dell&#39;ago in modo che la punta è adiacente alla regione anteriore del lato sinistro del torace della mosca. Portare sia la punta dell&#39;ago e del torace della mosca a fuoco con lo stereomicroscopio. </li><li> Spostare delicatamente il volo verso la punta dell&#39;ago in modo che la punta dell&#39;ago tocca il centro della protuberanza della regione prescutum sinistra del torace della mosca (Fig. 1). Una volta che la mosca è correttamente orientato e allineato con la punta dell&#39;ago, continuare ad avanzare al volo l&#39;ago in modo che l&#39;ago infilza il centro del prescutum. </li><li> Applicare una pressione positiva, se necessario facendo avanzare lo stantuffo della siringa con la manopola del micromanipolatore microinjector fino al 0,1 ml di liquido è stato iniettato nel volare. Il flusso del liquido in hemocoel della mosca impartirà un colore blu sul lato sinistro del torace anteriore. Ogni tanto si deve ritirare e avanzare più volte per rimuovere eventuali blocchi punta dell&#39;ago e consentire il flusso del fluido. </li><li> Lasciare vola iniettato per recuperare in fiale agar al 2% per i punti designati tempo prima dell&#39;estrazione emolinfa. </li></ol> 5. Emolinfa Collection <ol><li> Brevemente anestetizzare vola con CO 2 a umidificato desiderato post-iniezione punti ora e la posizione di ogni volo dorsale-side-down su nastro biadesivo apposte sulla superficie di un pad di CO 2. Disporre vola anche in filari con capsule testa allineati. Si può usare un bastoncino applicatore di legno tagliato a premere le loro ali nel nastro adesivo e garantire l&#39;immobilizzazione. </li><li> Una volta che le mosche sono fissati al nastro, afferrare proboscide ogni mosca con una pinza sottile e delicatamente tirare verso la regione ventrale e posteriore del volo in modo che la parte anteriore della capsula testa è visibile attraverso il microscopio stereo. </li><li> Con l&#39;altra mano e mentre si tiene la proboscide in posizione, forare il centro della capsula testa appena sopra la sutura ptilinal utilizzando un ago di tungsteno tagliente (Fig. 2). Bisogna stare attenti a non inserire l&#39;ago troppo lontano, come è facile forare la punta dell&#39;ago attraverso l&#39;altro lato della capsula testa e successivamente perdere controllo del flusso di emolinfa. Puntura vola tutti in un gruppo di iniezione prima di raccogliere emolinfa. </li><li> Una volta che la capsula di testa è stato perforato, utilizzare un bastone di legno tagliato applicatore per applicare delicatamente pressione sull&#39;addome della mosca. Può essere utile per lanciare i sorvolare in modo che poggi su un lato ventrale, come si applica la pressione. Questa procedura produrrà una goccia di emolinfa dal sito della puntura testa capsula. </li><li> Toccare un&#39;estremità di un tubo di 1ml microcapillare alla goccia emolinfa che emerge dal sito della puntura testa capsula. L&#39;emolinfa entra nel tubo attraverso l&#39;azione capillare. Determinare e registrare la quantità di emolinfa raccolti controllando la colonna di liquido all&#39;interno del tubo microcapillare contro un grafico del volume è laureato. </li></ol> 6. Emolinfa glucosio Determinazione <ol><li> Espellere i campioni emolinfa in pozzetti contenenti 100 ml di Reagente Glucosio Infinity. Tenere piastra su ghiaccio durante il caricamento di campioni emolinfa. </li><li> Stabilire una curva standard caricando separatamente 1 ml ciascuna delle soluzioni madre di glucosio a 50mm, 25mm, 12,5 mm, 6.25mm e 3.125mM. </li><li> Incubare i campioni a 37 ° C per 10 minuti. </li><li> Rileva a 340 nm. </li><li> Considerazione per il volume emolinfa raccolti e determinare la concentrazione di glucosio campione basato sulla curva standard con concentrazioni di glucosio noti. </li></ol> 7. Rappresentante Risultati Una tipica risposta di tolleranza insulinica viene rilevato nei diabetici insulino-iniettata vola in cui viene rilevato un calo dei livelli circolanti di glucosio 15 minuti dopo l&#39;iniezione. Al contrario, tale risposta non è visto nei moscerini della PBS iniettato (Fig. 3). Questa risposta in materia di smaltimento del glucosio periferico continua nei diabetici insulino-iniettata vola fino a 30 minuti dopo l&#39;iniezione. Noi abitualmente estrarre 0,2-0,5 ml di emolinfa per 4-5 vola in ogni gruppo di iniezione. Tre gruppi di iniezione sono inclusi in ciascun esperimento. <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="Figura 1"/> Figura 1. Lato sinistro del torace Drosophila che mostra l&#39;inserimento del sito ago (Modificato da Demerec, 1950) 7. Inserire l&#39;ago attraverso il centro della prescutum sul anteriore, regione dorsale del lato sinistro del torace. <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="Figura 2"/> Figura 2. Vista frontale della testa di Drosophila che mostra la collocazione puntura per l&#39;estrazione emolinfa (Modificato da Demerec, 1950) 7. Perforare la capsula testa con una sonda di tungsteno finemente incisi nel centro della capsula testa appena sopra la sutura ptilinal. <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="Figura 3"/> Figura 3. Una tipica risposta di tolleranza insulinica rilevato nel controllo delle mosche adulte. Controllo delle mosche w 1118 sono stati iniettati con insulina bovina (1 ng in PBS) o PBS solo. Vola in gruppi replicare sono stati poi permesso di recuperare per i minuti 0, 15 o 30 ed i livelli di glucosio circolanti sono stati misurati.

<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>

Nessun conflitto di interessi dichiarati.

La tecnica descritta in questo rapporto è potenzialmente utile in qualsiasi studio che indaga i processi fisiologici con conseguente alterazioni rilevabili nella composizione emolinfa Drosophila. Grazie alla combinazione di iniezione e la raccolta emolinfa in questo modo, è possibile accertare gli effetti immediati fisiologicamente rilevanti di un particolare trattamento sperimentale o manipolazione. Il vantaggio principale di questa tecnica &quot;salasso&quot; nella raccolta di emolinfa rispetto alle tecnich...

<table border="1"><tbody><tr><th> Nome del reagente / attrezzature </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Insulina bovina </td><td> Sigma </td><td> I5500 </td></tr><tr><td> Infinity glucosio reagente </td><td> Thermo Electron Corporation </td><td> TR1541 </td></tr><tr><td> Manuale microinjector </td><td> Sutter strumento </td><td></td></tr><tr><td> P-87 Flamming / Brown micropipetta estrattore </td><td> Sutter strumento </td><td></td></tr><tr><td> Singolo cilindro in vetro borosilicato capillare </td><td> Sistemi AM </td><td> 626000 </td></tr><tr><td> FD &amp; C blu n ° 1 </td><td> McCormick &amp; Company </td><td></td></tr><tr><td> 1 tubi microlitri microcapillare </td><td> Drummond </td><td></td></tr><tr><td> Tre assi micromanipolatore manuale e la base </td><td> Mondo Strumenti di precisione </td><td></td></tr></tbody></table>

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

Conservati i meccanismi di rilevazione dei nutrienti esistono tra di mammiferi e moscerino della frutta dove peptidi simili all&#39;insulina nei mammiferi e il glucagone, rispettivamente la funzione di mantenere l&#39;omeostasi del glucosio durante lo sviluppo larvale 1,2 fasi. Studi sulla gran parte post-mitotico mosche adulte hanno rivelato perturbazione dell&#39;omeostasi del glucosio come il risultato di ablazione genetica di insulin-like peptide (ILP), cellule che producono (IPC) 3. Così, moscerini della frutta adulti sono molto promettenti come un sistema idoneo modello genetico di malattie metaboliche tra cui diabete di tipo II. Per sviluppare ulteriormente il sistema di moscerino della frutta, paragonabile test fisiologici utilizzati per misurare la tolleranza al glucosio e la sensibilità all&#39;insulina nei mammiferi devono essere stabiliti. A tal fine, abbiamo recentemente descritto una nuova procedura per la misurazione della risposta orale di tolleranza al glucosio nei mosca adulta e ha dimostrato l&#39;importanza di IPC adulti nel mantenimento dell&#39;omeostasi del glucosio 4,5. Qui, abbiamo modificato la procedura descritta in precedenza per l&#39;iniezione di insulina 6 e combinato con un nuovo metodo di estrazione emolinfa per misurare la sensibilità periferica all&#39;insulina nei mosca adulta. Unicamente, il nostro protocollo permette misurazioni dirette fisiologica della capacità della mosca adulta di disporre di un carico di glucosio periferico a seguito di iniezione di insulina, una metodologia che rende possibile caratterizzare mutanti segnalazione dell&#39;insulina e gli interventi possibili riguardano tolleranza al glucosio e la sensibilità all&#39;insulina nei mosca adulta.

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

Segnalazione dell&#39;insulina conservato percorsi trova nel moscerino della frutta<em&gt; Drosophila melanogaster</em&gt; Fanno di questo organismo un potenziale strumento per la modellazione di malattie metaboliche tra cui diabete di tipo II. A tal fine, è fondamentale per stabilire i test fisiologici per misurare in modo efficace azione sistemica all&#39;insulina nello smaltimento del glucosio periferico nella mosca adulta.

L&#39;insulina per iniezione ed estrazione emolinfa per misurare la sensibilità all&#39;insulina nei adulti 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 Views.  State University of New York. Segnalazione dell'insulina conservato percorsi trova nel moscerino della frutta Drosophila melanogaster Fanno di questo organismo un potenziale strumento per la modellazione di malattie metaboliche tra cui diabete di tipo II. A tal fine, è fondamentale per stabilire i test fisiologici per misurare in modo efficace azione sistemica all'insulina nello smaltimento del glucosio periferico nella mosca adulta.

Video: 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.

Monitoraggio ad alta risoluzione video di locomozione per adulti in 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 ...

Ricerca

Biologia

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.

Dissezione di Oenocytes da adulti 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 dosaggio quantitativo di insulina che esprimono formante colonia Progenitori

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.

Misura della durata della vita in<em&gt; Drosophila melanogaster</em

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

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;utilizzo del Ramsay Assay misurare la secrezione di fluidi e Ion Flux Prezzi in<em&gt; Drosophila melanogaster</em&gt; Tubulo Malpighi

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.

Misurazione e risposta alla somministrazione di glucosio alla stimolazione dell&#39;insulina in<em&gt; In vitro</em&gt; Myotubes primario umano coltivato

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 Imaging di Neuropeptide Y-pHluorin: una tecnica per visualizzare esocitosi di granuli di insulina in isolotti intatti Murine ed umani

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.

Determinazione di zona ottico della sezione trasversale del muscolo dei muscoli adulto volo indiretto di 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.

Infezione batterica orale e spargimento 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.

Visualizzazione e monitoraggio di mRNA endogeni nelle camere uovo di Drosophila melanogaster dal vivo

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