Este trabalho foi financiado por concessões do NIA para YW.CF (AG21068, AG31086).

1. Preparação da solução de insulina <ol><li> Preparar a solução de insulina bovina fresca pela dissolução de insulina em PBS para atingir a concentração de 0,01 mg / ml. Tanto a insulina / PBS e soluções de controle PBS deve ser mantido no gelo durante todo o procedimento de injeção. Estas soluções devem ser preparadas com 0,5% (v / v) FD &amp; C no Blue. Um corante alimentar. </li></ol> 2. Preparação da agulha e injeção de Set-up <ol><li> Prepare agulhas capilar de vidro usando uma micropipeta puller.The seguintes configurações extrator produzir agulhas de qualidade suficiente para injecção: Calor, 345; Pull, 210; velocidade, 100; Time, 200 (100ms). Agulhas acabada de tirar deve ser anulado pela pressionar a ponta através de um tecido Kimwipe. Este processo remove embotamento da ponta alongada de alta resistência e produz uma ponta stouter com um diâmetro maior dos poros. </li><li> Lugar agulhas blunted ponta-up em um tubo de microcentrífuga contendo a insulina / PBS PBS solução ou sozinho. Capilar ação resulta em back-preenchimento de cada agulha. Observe a ponta da agulha sob um estereomicroscópio para garantir que nenhuma bolha de ar detritos ou aparecem na ponta. Descartar qualquer agulhas que não preencher de forma limpa. </li><li> Inserir agulhas cheias no suporte manual da agulha microinjetor e posicione o suporte da agulha com um micromanipulador de modo que a ponta da agulha é visível através de um estereomicroscópio. Aplique uma pressão positiva sobre a coluna de fluido na agulha girando o botão micrômetro manual de microinjetor ligado ao êmbolo da seringa microinjetor. </li><li> Verifique se pressão suficiente para o deslocamento de fluidos foi aplicada por tocar um Kimwipe para a ponta da agulha e confirmando o fluxo de fluido. </li><li> Uma vez que a agulha foi preparado para injeção, apor um gráfico de calibração para o eixo da agulha com fita adesiva transparente, e trazer a ponta da agulha em foco através de um microscópio estereoscópico com 20X de ampliação. </li></ol> 3. Fly Preparação <ol><li> Dez dias de idade moscas fêmeas são usados ​​para experimentos de injeção. Coletar moscas dentro de 24 h da eclosão. Anestesiar voa com CO 2 umidificado em uma almofada de gás, classificar para fora homens e moscas lugar feminino em frascos contendo dieta padrão ou tratamento. Manter as moscas fêmeas em dietas experimentais durante 10 dias. </li><li> No décimo dia após a eclosão e separação para dietas experimentais, a transferência de moscas de seu alimento contendo frascos para frascos contendo um plugue de 5 ml de agar 2% e privá-los por 12-16 h. </li><li> Transferência de moscas fome para frascos contendo filtros de glicose a 10% embebido por 1 hora antes da injeção de insulina. </li><li> Brevemente anestesiar as moscas com CO 2 umidificado após a ingestão de glicose e, em seguida imobilizá-los frio no gelo. </li></ol> 4. Procedimento de injeção <ol><li> Gentilmente segure uma mosca frio imobilizado com uma pinça fina e segurá-la perto da ponta da agulha para que a dica é adjacente à região anterior do lado esquerdo do tórax da mosca. Trazer tanto a ponta da agulha e do tórax da mosca em foco sob o estereomicroscópio. </li><li> Suavemente mova a voar em direção a ponta da agulha de modo que a ponta da agulha toca o centro da protuberância da região prescutum esquerdo do tórax da mosca (Fig. 1). Uma vez que a mosca é corretamente orientados e alinhados com a ponta da agulha, continuar a promover a voar para a agulha de modo que a agulha espeta o centro do prescutum. </li><li> Aplicar pressão positiva, conforme necessário, avançando o êmbolo da seringa com o botão do micromanipulador microinjetor de até 0,1 mL de fluido foi injetado na mosca. O fluxo de fluido em hemocele da mosca dará uma cor azul para o lado esquerdo do tórax anterior. Ocasionalmente é preciso retirar e avançar várias vezes para retirar qualquer bloqueio ponta da agulha e permitir o fluxo de fluido. </li><li> Permitir que voa injetado para recuperar em frascos de agar 2% para os pontos de tempo designados antes da extração hemolinfa. </li></ol> 5. Coleta de hemolinfa <ol><li> Brevemente anestesiar voa com CO 2 no umidificado desejado pontos após a injecção tempo e posição de cada lado da dorsal fly-down em fita dupla face afixada à superfície de um bloco de CO 2. Organizar voa no mesmo linhas com cápsulas cabeça alinhada. Pode-se usar uma vara de madeira recortada aplicador para pressionar as asas para a fita adesiva e garantir a imobilização. </li><li> Uma vez que as moscas são fixos para a fita, segure proboscis cada mosca com uma pinça fina e puxe-o cuidadosamente para a região ventral e posterior da mosca de modo que a frente da cápsula cefálica é visível através do microscópio estéreo. </li><li> Com a outra mão e, segurando a tromba na posição, fure o centro da cápsula cabeça logo acima da sutura ptilinal usando uma agulha de tungstênio afiadas (Fig. 2). É preciso ter cuidado para não inserir a agulha muito longe, pois é fácil dar um soco na ponta da agulha através do outro lado da cápsula cefálica e, posteriormente, perder control do fluxo de hemolinfa. Punção todas as moscas em um grupo de injeção antes de coletar hemolinfa. </li><li> Uma vez que a cápsula cefálica foi perfurado, use uma vara de madeira para aplicador aparadas aplique uma pressão suave abdômen da mosca. Pode ser útil para rolar a voar por cima de modo que repousa sobre o seu lado ventral como você aplicar pressão. Este procedimento irá produzir uma gota de hemolinfa a partir do sítio de punção cabeça cápsula. </li><li> Toque em uma extremidade de um tubo 1μl microcapilar para a gota de hemolinfa emergindo do local da punção cabeça cápsula. A hemolinfa entra no tubo através de uma acção capilar. Determinar e registrar a quantidade de hemolinfa coletadas, verificando a coluna de fluido no interior do tubo microcapilar contra um gráfico de volume graduado. </li></ol> 6. Determinação de glicose hemolinfa <ol><li> Eject amostras hemolinfa em poços contendo 100 ul do Infinito Reagente glicose. Manter placa no gelo durante o carregamento de amostras de hemolinfa. </li><li> Estabelecer uma curva padrão de carga separadamente cada 1 ml das soluções de reserva de glicose a 50mm, 25mm, 12.5mm, 6.25mM e 3.125mM. </li><li> Incubar as amostras a 37 ° C por 10 minutos. </li><li> Detectar absorvância a 340 nm. </li><li> Conta do volume de hemolinfa coletadas e determinar a concentração de glicose da amostra com base na curva padrão com concentrações de glicose conhecido. </li></ol> 7. Resultados representante Uma resposta típica de tolerância à insulina é detectado em insulina injetada moscas, onde uma queda nos níveis circulantes de glicose é detectado 15 minutos após a injecção. Em contraste, essa resposta não é visto em moscas PBS injetado (Fig. 3). Esta resposta na eliminação de glicose periférica continua em insulina injetada-moscas até 30 minutos após a injecção. Rotineiramente extrair 0,2-0,5 mL de hemolinfa por 4-5 moscas em cada grupo de injeção. Três grupos de injeção estão incluídos em cada experimento. <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="Figura 1"/> Figura 1. Lado esquerdo do tórax, mostrando Drosophila local da agulha de inserção (Modificado de Demerec, 1950) 7. Inserir a agulha através do centro da prescutum na região, anterior dorsal do lado esquerdo do tórax. <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="Figura 2"/> Figura 2. Vista frontal de Drosophila cabeça mostrando o local da punção por extração de hemolinfa (Modificado de Demerec, 1950) 7. Punção da cápsula cabeça com uma sonda de tungstênio finamente afiada no centro da cápsula cabeça logo acima da sutura ptilinal. <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="Figura 3"/> Figura 3. A resposta de tolerância à insulina típica detectada em adultos controle de moscas. Moscas controle w 1118 foram injetados com insulina bovina (1 ng em PBS) ou PBS apenas. Moscas em grupos replicar foram então autorizados a recuperar de 0, 15 ou 30 minutos e os níveis de glicose circulante foram medidos.

<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>

Não há conflitos de interesse declarados.

A técnica descrita neste relatório é potencialmente útil em qualquer estudo que investiga os processos fisiológicos, resultando em alterações detectáveis ​​em Drosophila composição hemolinfa. Ao combinar a injeção e coleta de hemolinfa desta forma, é possível verificar os efeitos imediatos fisiologicamente relevantes de um determinado tratamento experimental ou manipulação. A principal vantagem desta &quot;sangria&quot; técnica na coleta de hemolinfa em relação às técnicas anteriores envolvendo decapitação 2 é que esta técnica minimiza a contaminação das amostras de hemolinfa com cuidado contents.If intestino é tomado para não recolher qualquer tecido do corpo pericerebral gordura, que às vezes pode aparecer na exuded gota hemolinfa, em seguida, as amostras devem refletir com precisão o estado do aparelho circulatório em fluidos vivo. Um fator potencialmente de confusão inerente a qualquer experimento de injecção, realizado nessa escala é a diluição inicial de fluidos circulatória total após a injecção. Isto pode ser facilmente controlados, no entanto, injetando o mesmo volume de PBS em moscas experimental ou insulina em controles pareados por idade e normalizar os resultados. Para garantir reprodutíveis leituras de glicose em circulação, a qualidade de gotículas de hemolinfa é da maior importância. Apenas gotículas clara de hemolinfa desprovida de gordura corporal pericerebral ou restos de outros tecidos devem ser coletadas para a determinação de glicose. É também digno de nota que apenas até 8 amostras devem ser coletadas antes de determinar as concentrações de glicose relativa. Notamos uma &quot;deriva&quot; no OD 340 leituras quando as amostras de hemolinfa foram deixados no gelo por um período prolongado de tempo. Nosso protocolo deixa muito espaço para modificação com base em equipamentos disponíveis. Configurações extrator pipeta pode precisar ser modificado com base no modelo e condição do extrator. Nós descobrimos que as configurações adequadas para a produção de agulhas eletrodo intracelular de gravação são ideais para as agulhas de injeção. Além disso, enquanto nós empregou um micromanipulador de três eixos para manter a posição da agulha sob o estereoscópio, isso também pode ser conseguido com um ringstand resistente e sistema de fixação que a agulha permanece em uma posição fixa durante todo o procedimento de injeção. A alta resistência das agulhas usadas exigiu o desenvolvimento de um método alternativo para determinar o volume de fluido injetado como um movimento 01:01 êmbolo da seringa para o deslocamento de volume não foi possível. Nós desenvolvemos uma escala graduada que pode ser impresso e anexado ao lado do eixo da agulha. Dependendo do sistema utilizado, pode ser necessário para substituir algum fluido da agulha além do suporte da agulha do microinjetor de modo que o menisco líquido pode ser alinhada com o cartão de formaturas de calibração afixada ao lado do eixo da agulha. Finalmente, duas limitações desta técnica são notados em nosso protocolo. Primeiro, duas pessoas são geralmente necessários para coordenar a injeção e as etapas de extração de hemolinfa a fim de maximizar o número de amostras processadas. Em segundo lugar, as variações na resposta de tolerância à insulina são por vezes observadas no interior do mesmo genótipo. Acreditamos que este é devido a respostas variáveis ​​moscas indivíduo pode ter seguintes imobilização de gelo e de recuperação em agar. Portanto, um número suficiente de amostras deve ser examinado antes de conclusões confiáveis ​​podem ser tiradas.

<table border="1"><tbody><tr><th> Nome do reagente / equipamentos </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Insulina bovina </td><td> Sigma </td><td> I5500 </td></tr><tr><td> Infinito Reagente glicose </td><td> Thermo Electron Corporation </td><td> TR1541 </td></tr><tr><td> Manual microinjetor </td><td> Instrumento Sutter </td><td></td></tr><tr><td> P-87 Flamming / Brown micropipeta extrator </td><td> Instrumento Sutter </td><td></td></tr><tr><td> Único vidro de borosilicato barril capilar </td><td> Sistemas de AM </td><td> 626000 </td></tr><tr><td> FD &amp; C Azul No. 1 </td><td> McCormick &amp; Company </td><td></td></tr><tr><td> 1 ml tubos microcapilar </td><td> Drummond </td><td></td></tr><tr><td> Três eixos micromanipulador manual e base de </td><td> Precision Instruments mundo </td><td></td></tr></tbody></table>

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

Conservado nutrientes mecanismos de detecção existem entre mamíferos e mosca das frutas, onde os peptídeos semelhante à insulina e glucagon mamíferos, respectivamente a função de manter a homeostase da glicose durante o desenvolvimento 1,2 estágios larval. Estudos sobre moscas em grande parte pós-mitóticas adultos revelaram perturbação da homeostase da glicose como o resultado da ablação genética de insulin-like peptide (ILP), produzindo células (IPCs) 3. Assim, as moscas de fruta adultos são uma grande promessa como um sistema modelo adequado genética para doenças metabólicas, incluindo diabetes tipo II. Para aprofundar o sistema de mosca da fruta, comparável ensaios fisiológicos usados ​​para medir a tolerância à glicose ea sensibilidade à insulina em mamíferos deve ser estabelecida. Para este fim, temos recentemente descrito um procedimento novo para medir a resposta de tolerância oral à glicose na mosca adulta e demonstraram a importância de IPCs adulto na manutenção da homeostase da glicose 4,5. Aqui, temos um procedimento modificado descrito anteriormente para a injeção de insulina 6 e isso combinado com um novo método de extração hemolinfa para medir a sensibilidade periférica à insulina na mosca adulta. Excepcionalmente, o nosso protocolo permite medições fisiológicas direta da capacidade da mosca adulta de dispor de uma carga de glicose periférica após a injeção de insulina, uma metodologia que viabiliza a caracterizar mutantes sinalizadora de insulina e intervenções potenciais que afetam a tolerância à glicose ea sensibilidade à insulina na 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

Sinalização da insulina conservada caminhos encontrados na mosca da fruta<em&gt; Drosophila melanogaster</em&gt; Fazer este organismo uma ferramenta potencial para a modelagem de doenças metabólicas, incluindo diabetes tipo II. Para este fim, é fundamental para estabelecer ensaios fisiológicos para medir efetivamente a ação da insulina sistêmica na eliminação de glicose periférica na mosca adulta.

Injeção de insulina e Extração de hemolinfa para medir a sensibilidade à insulina em adultos 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

Nanyang Technological University

Research

JoVE Journal

Biology

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

19.6K visualizações.  State University of New York. Sinalização da insulina conservada caminhos encontrados na mosca da fruta Drosophila melanogaster Fazer este organismo uma ferramenta potencial para a modelagem de doenças metabólicas, incluindo diabetes tipo II. Para este fim, é fundamental para estabelecer ensaios fisiológicos para medir efetivamente a ação da insulina sistêmica na eliminação de glicose periférica na mosca adulta.

Vídeo: 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.

De alta resolução de vídeo de Rastreamento Locomotion em 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 ...

Biologia

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-orbital de injeção no Zebrafish Adulto

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

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.

Dissecção da Oenocytes de Adultos 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.

Uma análise quantitativa para expressar Insulin-formadoras de colônias Progenitores

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.

Medição do Tempo de vida em<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.

A utilização do Ramsay ensaio para medir secreção de fluidos e Ion Flux Preços no<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.

Medição da captação de glicose e resposta à estimulação de insulina em miotubos primários humanos cultivados in vitro

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 da imagem latente do Neuropeptide Y-pHluorin: uma técnica para visualizar a exocitose de grânulo de insulina em ilhotas murino e humanas intactas

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.

Determinação de área óptica transversal muscular dos músculos de voo indirecto adulto 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.

Infecção bacteriana e oral Shedding em 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 ...