这项工作是从NIA的YW.CF（AG21068，AG31086）的赠款支持。

1。胰岛素溶液的制备<ol><li>准备新鲜牛胰岛素溶液溶解在PBS中的胰岛素，达到0.01毫克/毫升的浓度。胰岛素/ PBS和控制PBS解决方案必须保持整个注射过程中的冰。这些解决方案应与0.5％（V / V）FD＆C蓝色没有准备。 1食品着色剂。 </li></ol> 2。针的制备和注射设置<ol><li>使用一个车夫设置产生足够的质量针注射微量puller.The：热火，345;拉，210;速度，100;时间，200（100毫秒），准备毛细玻璃针。刚拉的针，必须按提示通过Kimwipe组织减弱。这钝化过程中删除拉长的高抗尖和，产生一个具有较大孔径的stouter提示。 </li><li>将钝的针头，针尖在含有微量胰岛素/ PBS溶液或PBS管。在回填土每针毛细管行动的结果。观察，立体显微镜下，以确保无杂物或气泡出现在提示的针尖。放弃任何针头不干净填写。 </li><li>插入一个装满针的显微，使针尖通过立体显微镜下可见成手动微量持针器和位置的持针器。应用正压液柱针转动的手动微量微量注射器柱塞的千分尺旋钮。 </li><li>验证流体位移足够的压力，已应用于通过触摸一个Kimwipe针尖和确认流体流动。 </li><li>一旦针头已准备注射，贴上一个校准图表针轴与透明胶带，并带来20倍的放大倍率下通过体视显微镜的针尖成为焦点。 </li></ol> 3。粉煤灰制备<ol><li> 10天岁的雌性果蝇用于注射实验。羽化24小时内收集的苍蝇。麻醉与加湿的CO 2气体盘，整理出含有小瓶标准或治疗饮食的男性和雌性果蝇的地方苍蝇。维持10天的雌性果蝇实验的饮食。 </li><li>在羽化和分离实验的饮食后的第十天，转让苍蝇从他们的食物含瓶小瓶5毫升含有2％的琼脂插件，饿死他们为12-16小时。 </li><li>小瓶含10％葡萄糖注射胰岛素前1小时浸泡过滤器传输饿死的苍蝇。 </li><li>简言之麻醉与加湿的CO 2的苍蝇葡萄糖喂养后，再用冷水固定在冰上。 </li></ol> 4。注射过程<ol><li>轻轻抓住冷固飞与一双细镊子，并保持它靠近针尖，使针尖飞的胸部左侧前地区毗邻。纳入重点下的体视显微镜的针尖和果蝇的胸部。 </li><li>轻轻移动飞朝针尖，使针尖触及左飞的胸部（图1）prescutum地区突起的中心。一旦飞是正确导向，对准针尖，继续推进到飞针，使针头刺穿prescutum中心。 </li><li>应用积极的压力，需要通过推进注射器柱塞微量的显微操纵器旋钮，直到成飞已注入0.1μL液。成飞的hemocoel流体的流动将传授蓝色前胸部的左侧。有时，必须收回，提前多次驱逐任何针头堵塞，使流体流动。 </li><li>允许注入果蝇血淋巴中提取前指定的时间点恢复2％琼脂小瓶。 </li></ol> 5。淋巴收集<ol><li>简要麻醉与加湿的CO 2所需的注射后的时间点和位置苍蝇每飞，背端的CO 2垫表面贴双面胶带。安排在偶数行头对齐胶囊苍蝇。我们可以使用一个镶着木涂药棒到磁带粘合剂按它们的翅膀，并确保固定。 </li><li>一旦苍蝇固定到磁带上，把握每一个飞行的长鼻，用细镊子，轻轻一拉，对腹，然后后区飞使头舱的前方是通过立体显微镜可见。 </li><li>用另一只手按住的长鼻位置，穿刺中心的正上方ptilinal缝合头部胶囊，使用锋利的钨针（图2）。我们必须注意不要插入针太远，因为它很容易打穿头舱的另一侧针尖，随后失去彗星ontrol流的血淋巴。穿刺所有苍蝇之前收集血淋巴注射组。 </li><li>一旦被刺破头囊，使用修整木涂药棒轻轻施加压力，以苍蝇的腹部。它推出过这样的飞行，它在其腹侧在于你施加压力可能会有所帮助。此过程会产生从头部囊穿刺部位的血淋巴中的液滴。 </li><li>触摸一个加入1μlmicrocapillary管从头部囊穿刺部位出现血淋巴液滴的一端。将通过毛细作用进入血淋巴管。确定并记录通过检查microcapillary管内液柱对一个毕业于量图表收集血淋巴量。 </li></ol> 6。淋巴葡萄糖测定<ol><li>弹出到微孔板含100μL无限葡萄糖试剂的血淋巴样品。板，而加载血淋巴样品置于冰上。 </li><li> 50MM，25MM，12.5MM，6.25mM和3.125mM分别装载葡萄糖储备液各1μL，建立标准曲线。 </li><li>孵育样品在37 ° C下10分钟。 </li><li>检测在340 nm处的吸光度。 </li><li>收集血淋巴量的帐户，并确定样品中葡萄糖浓度的基础上，与已知的血糖浓度的标准曲线。 </li></ol> 7。代表性的成果一个典型的胰岛素耐受反应检测胰岛素注射循环中的葡萄糖水平下降是检测到注射后15分钟的苍蝇。相比之下，这样的反应是没有看到在PBS注入果蝇（图3）。飞起来，注射后30分钟，这在周边葡萄糖处置的响应，继续在胰岛素注射。我们经常提取每4-5苍蝇在每次注射组0.2-0.5微升血淋巴。三注射组包括在每一个实验。 <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="图1"/> 图1。果蝇表现出针插入部位（从Demerec，1950年修订）7胸部的左侧。通过prescutum前，左侧胸廓的背地区的中心插入针。 <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="图2"/> 图2显示穿刺血淋巴中提取的位置（从Demerec，1950年修订）7的果蝇的正面观。刺头细削尖的钨探针在中心的正上方ptilinal缝合头胶囊胶囊。 <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="图3"/> 图3，一个典型的胰岛素耐受反应检测控制成人苍蝇。 1118苍蝇控制瓦特注射牛胰岛素的PBS（1毫微克）或PBS。复制组中的苍蝇，然后让其恢复为0，15或30分钟，循环血糖水平进行了测量。

<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>

在本报告中描述的技术在任何调查在果蝇血淋巴成分的检测的改变导致生理过程的研究，是潜在有用的。通过以这种方式相结合的注射和血淋巴收集，它是可能的，以确定一个特定的实验性治疗或操纵的切身有关生理影响。这种“放血”技术的主要优点是收集超过以前的技 ​​术，涉及斩首 2血淋巴，这种技术最大限度地减少污染血淋巴样品与肠道contents.If护理是不收取任何pericereb...

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th></tr><tr><td>牛胰岛素</td><td>西格玛</td><td> I5500 </td></tr><tr><td>无限葡萄糖试剂</td><td>热电公司</td><td> TR1541 </td></tr><tr><td>手动微量</td><td>萨特仪器</td><td></td></tr><tr><td> P - 87 Flamming /布朗微量拉马</td><td>萨特仪器</td><td></td></tr><tr><td>单桶毛细管硼硅玻璃</td><td>上午系统</td><td> 626000 </td></tr><tr><td> FD＆C蓝色1号</td><td>的McCormick＆公司</td><td></td></tr><tr><td> 1μLmicrocapillary管</td><td>德拉蒙德</td><td></td></tr><tr><td>三轴手动显微操纵器和基</td><td>世界精密仪器</td><td></td></tr></tbody></table>

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

保守的营养感应机制之间存在着哺乳动物和果蝇肽类似哺乳动物的胰岛素和胰高血糖素，分别在幼虫阶段发育1,2维持血糖平衡的功能。主要有丝分裂后的成年果蝇的研究发现扰动糖代谢的胰岛素生产细胞（IPCS）3样肽（ILP）的遗传消融的结果。因此，成年果蝇保持一个合适的代谢紊乱，包括II型糖尿病的遗传模型系统的巨大潜力。为了进一步发展果蝇系统，用于测量哺乳动物糖耐量和胰岛素敏感性的可比生理检测，必须建立。为此，我们最近测量口服葡萄糖容忍的反应在成人飞介绍一种新的程序，并表现出成人的IPC的重要性，在维持血糖稳态4,5。在这里，我们已经修改为先前所描述的胰岛素注射6的程序，并结合血淋巴中提取了一种新的方法来测量在成人飞周围胰岛素敏感性。与众不同的是，我们的协议允许成年果蝇的能力，处置周边葡萄糖负荷时注射胰岛素的方法，使胰岛素信号突变和影响成人飞糖耐量和胰岛素敏感性的潜在干预是可行的特点直接的生理测量。

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

保守的胰岛素信号通路在果蝇中发现<em&gt;果蝇</em&gt;使这个有机体建模，包括II型糖尿病的代谢紊乱的潜在工具。为此，关键是建立生理检测在成人飞周边葡萄糖处置全身胰岛素的作用，以有效衡量。

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</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 次观看.  State University of New York. 保守的胰岛素信号通路在果蝇中发现果蝇使这个有机体建模，包括II型糖尿病的代谢紊乱的潜在工具。为此，关键是建立生理检测在成人飞周边葡萄糖处置全身胰岛素的作用，以有效衡量。

视频: 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.

高分辨率视频跟踪移动的，在成年果蝇。

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

科研

生物学

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.

从成人Oenocytes剖析果蝇</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.

一个定量测定胰岛素表达细胞集落形成先觉

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.

寿命测量<em&gt;果蝇</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.

使用拉姆齐试验来测量液体分泌和离子流率在<em&gt;果蝇</em&gt;马氏管

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.

葡萄糖摄取测量和对胰岛素刺激的反应<em&gt;体外</em&gt;培养的人类初级肌管

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.

神经肽 Y-pHluorin 的共焦成像: 一种完整的小鼠和人胰岛胰岛素颗粒胞的可视化技术

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.

果蝇成人间接飞行肌的光学横截肌面积测定

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.

果蝇的口腔细菌感染和脱落

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.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

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