Name: a buoyancy-based method of determining fat levels in drosophila
Uploaded: 2016-11-01T11:15:00
Description: Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

K.E.H. was supported by the Training Grant in Molecular Biology NIH-T32-GM08730. This work was supported by NIH, NIDDK Grant 5K01DK095932 and AHA Award 12BGIA11930014 to T.R.

1.苍蝇利益的基因型收集鸡蛋注：理想的十字架包括150处女蝇和至少75男性。股票集合应该包含至少200苍蝇。 <ol><li>准备葡萄板。 <ol><li> 37.5克琼脂添加到1.5升蒸馏水在一个4升烧瓶中。 </li><li>高压灭菌水/琼脂混合在121°C（250°F）50分钟。 </li><li>虽然水/琼脂的搭配是高压灭菌50克蔗糖添加至500ml葡萄汁，并在加热搅拌板上搅拌棒搅拌，直至蔗糖溶解。 </li><li>关火，继续搅拌至葡萄汁/蔗糖混?...

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有很多已研究出一种测定脂质水平8技术众多- 10。但是，上述每种方法带有由以上概述的基于浮力的测定法解决了一些重大的缺点。首先，这个实验非常快。测试一个完整的浓度梯度时间不超过30 - 60分钟。这是对目前使用的大部分技术的巨大改善。例如，通过质谱脂质定量需要7 - 9小时以分离脂质和另一个几个小时来分析它们。这是一个强大的威慑力上的大量动物进行?...

果蝇已遗传学等基本生物学问题的研究中已经使用了一个多世纪。在过去的几十年中，已经变得清楚的是果蝇是在许多人类疾病的研究的强有力的工具。由于70 -与人类疾病相关的基因有80％有确定的飞行直系同源基因1 - 4， 果蝇提供来研究复杂疾病的简化还没有翻译系统。代谢尤其是从这样的研究中受益。不仅是新陈代谢的苍蝇和人类之间高度保守的基因，但有关机关和细胞类型也非常相似2,5。例如，苍蝇同时存储三酰基甘油酯（TAG）和糖原，功能类似于在哺乳动物肝脏和白色脂肪组织6中执行的脂肪体。用果蝇作为人类肥胖的模式已经大大提高了我们的脂质的理解代谢和肥胖7的遗传学。发展的幼虫阶段为，因为它是专门用于饲养pupariation期间使用能量的存储学习营养素存储或利用的偏析特别有用。 目前，在果蝇确定脂肪储存水平的许多不同的定量方法。最广泛使用的方法是耦合比色测定（CCA）8,9。 CCA被用于确定人血清中的TAG水平制定并在该甘油从甘油三酯的骨架中释放将发生一些反应，最终导致在氧化还原偶联反应产生有色产物的前提进行操作。然后光的特定波长的吸光度测定来确定本甘油的初始量。然而，甘油，也可以从单 - 和二酰基甘油酯除了TAG解放，因此可能无法为s的精确测量灌装液位体脂肪9。此外，粉碎成蝇眼色素可以与一些吸光度读数干扰和结果9,10复杂化。因此，CCA必须伴随并通过薄层色谱（TLC），其允许可通过光密度10,11来定量最脂质家庭分离验证。然而，一些脂质类，如甾醇不能被分析和必须量化以不同的方式12。质谱（MS）是定量主要细胞脂质12,13的所有类的精确方式。然而，MS分析所需要的脂类提取程序都是耗时（多数回吐将近一整天），成本高。在这里，我们提出了一个替代方法快速，廉价地确定在果蝇的幼虫L3生物体的脂肪含量。 下面提出的方法利用在脂肪组织和瘦组织之间的密度差。哺乳动物发吨组织具有大约为0.9g / ml的14而骨骼肌的密度为1.06克/毫升15的密度。这种差异意味着，动物脂肪较高的卖场会比动物脂肪较低的门店，这将让他们在固定密度的解决方案，更好的浮动密度较低。此属性，可大量而被价格便宜，非侵入性动物的极快的筛选。基于浮力分析已经同时用于确认改变脂肪水平的公知的调节剂，以及识别肥胖16,17的新的基因和神经调节的表型。

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			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
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			<td height="42" style="height:42px;width:216px;">Welch&#39;s Original 100% Grape Juice</td>
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			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tegosept</td>
			<td style="width:168px;">Genesee Scientific</td>
			<td style="width:119px;">20-259</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
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			<td>Enriched degerminated</td>
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			<td height="21" style="height:21px;width:216px;">Drosophila Agar Type II</td>
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			<td height="21" style="height:21px;width:216px;">Propionic Acid</td>
			<td>VWR</td>
			<td>U330-09</td>
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			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
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			<td height="21" style="height:21px;">Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655-100G</td>
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			<td height="21" style="height:21px;width:216px;">Sodium phosphate anhydrous</td>
			<td style="width:168px;">Fisher</td>
			<td style="width:119px;">S3933</td>
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			<td height="21" style="height:21px;">Potassium chloride</td>
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			<td>P330-500</td>
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			<td height="21" style="height:21px;width:216px;">35 x 10mm petri plate</td>
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			<td height="21" style="height:21px;width:216px;">50mL Conical</td>
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			<td style="width:119px;">50-869-569</td>
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a buoyancy-based method of determining fat levels in drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

图1表示了如何基于浮力的测定可以在遗传操作的幼虫检测更高和更低的脂肪储存，例如， 图1A示出神经元的某些子集（E347）的幼虫大脑激励或沉默诱导较低和较高的脂肪分别商店。相同的遗传背景对照是在这两种情况下使用。 图1B提供了如何通过密度测定法获得的这种表型是由中性脂质提取和定量通过GCMS证实一个例子。 <p class="...

Watch this Scientific Journal Video about A Buoyancy-based Method of Determining Fat Levels in Drosophila at JoVE.com

A Buoyancy-based Method of Determining Fat Levels in Drosophila

这里，我们提出以测量在第三龄（L3） 果蝇幼虫阶段有机体脂肪含量的方法。这种方法利用脂肪组织相对较低的密度与改变脂肪储存幼虫区分。基于浮力分析是快速，重现性好，经济的筛选有价值的工具。

确定脂肪水平在基于浮力法<em&gt;果蝇</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...

Research

JoVE Journal

Biology

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Studying Aggression in Drosophila (fruit flies)

Studying Aggression in Drosophila fruit flies

学习在果蝇的侵略（果蝇）

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

科研

生物学

Pyrosequencing: A Simple Method for Accurate Genotyping

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Visually Mediated Odor Tracking During Flight in Drosophila

在果蝇的飞行过程中可视介导的气味追踪

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

In-vivo Centrifugation of Drosophila Embryos

In-vivo Centrifugation of Drosophila Embryos

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Heme Synthesis Levels in Mammalian Cells

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

的透的一种方法<em&gt;果蝇</em&gt;胚胎用于小分子活性的测定

The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

确定膜蛋白拓扑使用荧光蛋白酶保护（FPP）

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...