Name: measurement of lifespan in drosophila melanogaster
Uploaded: 2013-01-07T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Lifespan in Drosophila melanogaster at JoVE.com

这项工作是支持的资金从埃利森医学基金会（SDP， <a href="http://www.ellisonfoundation.org/index.jsp" target="_blank">http://www.ellisonfoundation.org/index.jsp</a> ），美国国立卫生研究院K01AG031917（NJL <a href="http://www.nih.gov" target="_blank">http://www.nih.gov/</a> ），美国国立卫生研究院5T32GM007315-35 （JR）和NIH R01AG030593（SDP）。这项工作利用的资源对果蝇衰老的核心弥敦道冲击卓越中心（DAC）的生物学老化的老化P30-AG-013283（ <a href="http://www.nih.gov" target="_blank">http://www.nih.gov/</a> ）由国家资助。作者想感谢的的普莱彻实验室为有益的讨论，特别是布赖恩涌的批判性阅读的手稿。我们要感谢尼克·阿谢尔和凯瑟琳Borowicz与数据采集的援助。

我们建议存储实验食品，酵母膏，和葡萄的琼脂平皿上，出现在协议中，在4℃下，在1-2个月内，并使用它们，只要模具和干燥设置不英寸的标准环境条件下的幼虫和成虫阶段涉及维护一个12:12的人力资源光线暗的周期和60％的相对湿度在25°C的苍蝇孵化器。 1。制备实验性食品的<ol><li>幼虫的生长，我们使用修改后的加州理工学院，这是简称为CT在本协议中的中等5。 <...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifespan+and+reproduction+in+Drosophila:+New+insights+from+nutritional+geometry.">Lifespan and reproduction in Drosophila: New insights from nutritional geometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 2498-2503 (2008).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental gerontology. 40, 386-395 (2005).</li></ol>

这里提出的协议描述了一种方法，在果蝇成虫寿命产生可重复的测量，是适应评估遗传，药理，和环境干预。该协议的关键方面包括仔细控制幼虫发育环境，最大限度地减少成人的压力，并尽量减少在实验组和对照组的偏见。我们还提出了使用的dLife寿命实验管理软件。通过简单的连接条形码或RFID标签，每个小瓶中，dLife计划将有助于为每个测量数据采集和绘制存活曲线。虽然它是目前最?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>活性干酵母</td><td> Fleishmann的酵母</td><td> 2192 </td><td></td></tr><tr><td>葡萄琼脂粉预混料</td><td>杰纳西科学</td><td> 47-102 </td><td></td></tr><tr><td>大胚胎回收笼</td><td>杰纳西科学</td><td> 59-101 </td><td></td></tr><tr><td>大更换端盖</td><td>杰纳西科学</td><td> 59-103 </td><td></td></tr><tr><td> 6盎司方底瓶，聚丙烯</td><td>杰纳西科学</td><td> 32-130 </td><td></td></tr><tr><td> Flugs封闭储存瓶</td><td>杰纳西科学</td><td> 49-100 </td><td></td></tr><tr><td> 果蝇样品瓶，宽，聚苯乙烯</td><td>杰纳西科学</td><td> 32-117 </td><td></td></tr><tr><td> Flugs封宽瓶</td><td>杰纳西科学</td><td> 49-101 </td><td></td></tr><tr><td>宽孔径的土豚枪头，200微升</td><td>丹维尔科学</td><td> P1105-CP </td><td></td></tr><tr><td> Flystuff Flypad，标准尺寸</td><td>杰纳西科学</td><td> 59-114 </td><td></td></tr><tr><td> BD猎鹰15毫升锥形离心管</td><td> Fisher Scientific则</td><td> 14-959-70C </td><td></td></tr><tr><td> Fisherbrand培养皿清除盖凸状; 100（外径）×15 mm（高）; </td><td> Fisher Scientific则</td><td> 08-757-12 </td><td></td></tr><tr><td> Kimax Colorware瓶1000毫升黄色</td><td> Fisher Scientific则</td><td> 10-200-47 </td><td></td></tr><tr><td> PBS pH值7.4的10倍</td><td>在vitrogen </td><td> 70011044 </td><td></td></tr><tr><td>石花菜琼脂</td><td> Mooragar </td><td> N / A </td><td></td></tr><tr><td>啤酒酵母</td><td> MP Biomedicals </td><td> 0290331280 </td><td></td></tr><tr><td>砂糖</td><td>克罗格（Kroger） </td><td> N / A </td><td></td></tr><tr><td> Tegosept </td><td>杰纳西科学</td><td> 20-266 </td><td>飞食品防腐剂</td></tr><tr><td>丙酸，99％ </td><td> Acros Organics公司</td><td> 149300025 </td><td>飞食品防腐剂</td></tr><tr><td>硫酸卡那霉素</td><td> ISC BioExpress </td><td> 0408-​​10G </td><td></td></tr><tr><td>盐酸四环素</td><td> VWR </td><td> 80058-724 </td><td></td></tr></tbody></table>

measurement of lifespan in drosophila melanogaster

老龄化是一个现象，就是在稳定的生理恶化，几乎所有的生物体中，它已审查的结果，减少物理性能和疾病的风险增加。个人老龄化是明显的年龄相关的死亡率增加，这往往是在实验室中测量的观测寿命在大样本的年龄相匹配的个人在群体水平上。实验，寻求量化的程度，遗传或环境的操作影响在简单的模型生物的寿命已经非常成功的理解方面的老化，延长寿命，预防与年龄有关的疾病在哺乳动物中是保守的类群和鼓舞人心的新战略。 飞醋， 黑腹果蝇（Drosophila melanogaster），是一个有吸引力老化的机制，由于其寿命相对较短，畜牧业方便，轻便遗传学研究的模式生物。然而，人口老龄化的措施，包括年龄特异性生存率和死亡率，即使是轻微的实验设计及环境的变化是非常敏感的，需要维护的持续时间老化实验，严格的实验室操作规范。这些考虑，一起实践需要仔细控制的遗传背景，用于产生强大的测量是必要的。事实上，有许多著名的推论寿命实验，酵母，蠕虫，果蝇和小鼠已追踪到环境或遗传的文物1-4的争议。在这个协议中，我们描述了一系列的程序进行了优化，经过多年的测量长寿果蝇的实验室瓶。我们还描述了使用的dLife软件，这是由本实验室开发的，并且是可用于下载（ <a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/ple</a>tcherlab /软件）。 dLife，加速吞吐量和促进良好做法，通过整合优化的实验设计，简化飞处理和数据采集，规范数据分析。我们还将讨论很多潜在的隐患在设计，收集和解释的寿命数据，我们提供的步骤，以避免这些危险。

在图1中，列出关键步骤的简化方案的协议。同步协议的一部分，可用于各种检测，要求年龄匹配的成年家蝇。 野生型苍蝇的在图2a中所示的典型的存活曲线，使用dLife实验管理软件（ 图2b，三 ）。成年男性通常居住的时间较短，与这两个群体实现对10％SY食品的寿命&gt; 50天的均值和中位数在25°C需要注意的是生存在早期的实验的一部分仍?...

Watch this Scientific Journal Video about Measurement of Lifespan in Drosophila melanogaster at JoVE.com

Measurement of Lifespan in Drosophila melanogaster

黑腹果蝇（Drosophila melanogaster）是一个功能强大的模式生物，探索长寿调控的分子基础。该协议将讨论生成一个以人群为基础的重现性好，测量的长寿以及潜在的缺陷和如何避免它们所涉及的步骤。

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 ...

measurement-of-lifespan-in-drosophila-melanogaster

Research

JoVE Journal

Biology

Measurement of Lifespan in Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

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 ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

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 ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

通过影像学的蛹壳<em&gt;果蝇</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

增强Northern印迹检测小RNA种类的<em&gt;果蝇</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

从放射性标记的底物在二氧化碳生成的测量<em&gt;果蝇</em

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human ...