This work was supported by Adelphi University faculty development grant and grant R15GM113156 from NIGMS awarded to EVC, grant R01GM067862 from NIGMS and grant R01AG027849 from NIA awarded to DMR.

1.试剂及解决方案<ol><li>对飞食品和媒体准备<ol><li>热蝇成分11％的糖，2％自溶酵母，5.2％玉米粉，琼脂0.79％w / v的水在加热板设定为90℃。经常搅拌，直到得到均匀稍密混合物。准备的550毫升飞食品的终体积为总共100小瓶用5mL飞食品中的每个。 </li><li>从加热源中删除，偶尔搅拌直到食物冷却到80℃并加入0.2％tegosept-4-羟基苯甲酸甲酯溶解在95％的乙醇。 </li><li>在两个相等的体积分割的食物。添加1.1的50mM的雷帕霉素溶解在乙醇中以一个卷毫升和1.1毫升乙醇的其他体积。搅拌雷帕霉素和乙醇溶液进入食品。可以肯定的温度加入药物前降低至50℃。 </li></ol></li><li>隔离缓冲<ol><li>制成500毫升之分离缓冲液[225 mM的甘露醇，75mM的蔗糖，10毫3-（N- &lt;的/ em&gt;的吗啉代）丙磺酸（MOPS）和1mM乙二胺四乙酸（EDTA），2.5毫克/毫升牛血清白蛋白（BSA）]中的水。 </li><li>用氢氧化钠将pH调节至7.2。初始pH将是酸性的。 </li><li>用无菌过滤器存储瓶用0.22μm孔径消毒溶液并储存在4℃。 </li></ol></li><li>洗涤液<ol><li>制成500毫升之洗涤缓冲液[225 mM的甘露醇，75mM的蔗糖，10毫米氯化钾，10毫摩尔Tris HCl和5mM的KH 2 PO 4]水。 </li><li>用氢氧化钠将pH调节至7.2。初始pH将是酸性的。 </li><li>用无菌过滤器存储瓶用0.22μm孔径消毒溶液并储存在4℃。 </li></ol></li></ol> 2.饲养果蝇品系<ol><li>在开发过程中控制幼虫密度，放置在一个培养瓶25对的父母，让他们产卵48小时。 </li> &lt;LI&gt; 48小时后取出的父母来调节蛋密度。 </li><li>利用新兴的F1后代重复步骤2.1和2.2，因此两代这个浓度控制的实现。 </li><li>为了收集大人，麻醉使用二氧化碳（CO 2）和独立的性别苍蝇。 <ol><li>使用单级调节器提供纯净的二氧化碳从高压力罐在5磅的连续流。防止静电，使用塑料管以催化二氧化碳到500ml过滤烧瓶中的水。 </li><li>用橡胶塞与一个孔以密封烧瓶。使用塑料管向烧瓶的侧孔连接到的 CO 2垫。 </li><li>将苍蝇在垫为不超过10-15分钟以上。允许从麻醉放置在实验条件下才恢复24小时。 </li></ol></li><li>为了产生足够的代谢产物线粒体丰富分数，使用300苍蝇每个样品。在这里，用女性，但gendeR可以不同的其它实验。转移150苍蝇自制1L的人口笼。允许访问5毫升飞食品在小瓶中。 </li><li>转移剩余的150苍蝇另一1升笼子。不要overpopulate笼150多个苍蝇。 </li><li>使用每个实验条件下六个复制样本。由于整个动物提取物产生更多的代谢产物，产生整个动物的1个样本分离，转移50名女飞行一个笼子里。作为线粒体分离物，使用每个实验条件6重复样品。 </li><li>地方笼在25℃为12小时光照和12小时黑暗周期。 </li><li>提供新鲜食物，每2或3天在小瓶中，用5ml的食物，以保持食品质量。 </li><li>保持苍蝇在食物10天。这个步骤是需要的雷帕霉素治疗7。其他治疗方法或条件会有所不同。 </li></ol> 3.隔离线粒体馏分<ol><li>转储飞从一个笼子（150苍蝇）成玻璃 - 聚四氟乙烯Dounce匀浆装入1ml冷冻隔离缓冲液中。 </li><li>通过移动杵上下15招均质。置于冰上砂浆，以保持线粒体完好无损。 </li><li>转移匀浆1.5ml试管并离心，在300×g离心5分钟，在4℃。 </li><li>取上清和离心6000 xg离心10分钟，在4℃下以富集线粒体。 </li><li>作为上清含有细胞质部分，丢弃上清液或将其用于替代实验。重悬在300微升的洗涤缓冲液的沉淀。 </li><li>重复步骤3.1 - 3.5第二个笼子。结合这两个笼子里的悬浮颗粒在低温离心管中。 </li><li>离心机在6000×g离心在4℃下10分钟。 </li><li>弃上清，闪冻在液氮中沉淀。 </li><li>存储线粒体富集的馏分在-80℃或更低的温度。 <ol><li>或者，对于整个动物预paration，将成年人在低温离心管中。闪光冻结液氮和存储成年人小瓶在-80℃或更低的温度。 </li></ol></li></ol>

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</ol>

作者什么都没有透露。

在这个协议中最关键的步骤是:1）足够的饲养苍蝇充裕的空间。这是非常重要的不overpopulate的人口笼150多个苍蝇每一个; 2）改变笼经常避免食物竞争和营养应激的食物;和3）保持所有样品在4℃的线粒体级分的分离过程中，以确保完整性。此外，还建议以冷却该隔离缓冲器，洗涤缓冲液，并在使用前的玻璃 - 聚四氟乙烯Dounce匀浆。为了减少富集线粒体派别胞浆污染，从步骤3.5线粒体颗粒可以用洗涤缓冲液。 从丰富的线粒体部分代谢产物的提取，需要大量的重复样品（6次重复/实验条件下）的。总共，24网箱用于图1和2。所以我们建议所有S的提取amples当天以降低实验的可变性。虽然可行，此协议限嗣继承精心的策划和准备所需的时间提前材料。标注时间提前管，并设计一个计划，以确保样本间的等待时间的一致性是需要保存样品的质量和重复的减少变化。 通常用于提取线粒体缓冲器含有糖10,11;因此，特别是糖代谢物的分析可能受的提取方法。虽然我们还没有使用他们在我们的分析，氯化钾提取缓冲区可替代线粒体隔离缓冲区含有糖12。 由于代谢网络的稳态性质和其介导此反应的蜂窝信号一个复杂的系统，改变线粒体代谢可以通过在全细胞代谢物池​​移位进行检测。在和线粒体的情况下这种系统的里亚尔调节，细微的代谢物的变化可能受到阻碍，除非足够的代谢物从线粒体丰富分数提取。在模型系统中大量的线粒体可以从组织中分离，分辨率高代谢成功地探测到的变化线粒体表型13。然而，在果蝇 ，窝藏足够质量的代谢物的样品的良好组合物可以是一个挑战。使用这个协议中，我们获得了高数量，质量和品种的代谢产物，证明不同的提取协议显示不同的代谢物的变化对整个飞与线粒体分数。虽然线粒体和细胞质蛋白的Western印迹表明蛋白质的小污染在两个级分，确认线粒体代谢物，附加步骤详细定量测量，以限制胞质代谢物的污染，在线粒体级分可能是申请IED。冷冻样品和这些代谢物的那些的冷冻/融化步骤后获得的比较之前代谢物的测量可以提供线粒体特定代谢物的一个更准确的定量。然而，在该模型，其中我们操纵线粒体基因组时，程序我们已用来制备样品是特别的信息和可能指向代谢重编程的通过线粒体介导的新颖途径。

此过程的目标是开发能产生足够的线粒体代谢物用于使用果蝇代谢组学研究富集线粒体级分。根据我们的经验，采用全细胞提取方法代谢组学分析是无法检测果蝇微妙的线粒体代谢物的变化。然而，线粒体分馏之前代谢分析增加了灵敏度，以确定线粒体代谢物变化。 线粒体是负责提供90％的细胞所需的正常功能1的能量的细胞器。在最近几年，已经认识到，线粒体在细胞和生物体功能的更加动态的作用不仅仅是产生三磷酸腺苷（ATP）和现在被确认为枢纽的代谢平衡2,3的调节。线粒体是一种共生的过程中，存款保险计划的结果tinct微生物系1.5十亿年前4合并〜。由于线粒体演变成真正的细胞器，基因的共生细菌在新兴的核基因组被合并。在动物今天，约1500线粒体蛋白是核编码的，而37个基因保留在线粒体DNA，其中13个编码线粒体蛋白质，是氧化磷酸化5的酶复合物的亚基。线粒体和核车厢之间的协调是必要的，以保持适当的线粒体功能。 使用此处描述的方法，我们能够探测在果蝇线粒体代谢变化所造成的操纵线粒体和核基因组之间的协调。我们用果蝇的菌株，其中线粒体DNA，从它的姊妹品种D. simulans置于单个D.果蝇核背景6。这种"破坏"mitonuclear基因型相比D的 "天然"或共同进化mitonuclear基因型果蝇携带着其原生四相同的核基因组果蝇线粒体DNA。该D.果蝇和D. simulans线粒体DNA相差〜100个氨基酸和影响mitonuclear通信7,8&gt; 500同义替换。我们生成的整个飞行提取物和线粒体富集提取物，研究代谢物的变化响应的药理压力。在这里，我们表明，使用线粒体富集的级分，当我们检测在本机，共同进化基因型携带D之间线粒体代谢物显着的变化果蝇线粒体DNA和破坏携带型D. simulans线粒体DNA。与此相反，这两种基因型之间的代谢变化是细微的使用，利用全动态提取正常的方法。因此，这种方法提供了一种路径理解线粒体DNA如何介导线粒体的变化响应于不同的药物。

<table><tbody><tr><td>0.2% tegosept-methyl 4-hydroxybenzoate</td><td>VWR</td><td>AAA14289</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>792799</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino) propanesulfonic acid (MOPS)</td><td>Sigma-Aldrich</td><td>M1254&amp;nbsp;</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>38057&amp;nbsp;</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>5470</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>RES3098T-B7</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1551139</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pads to anesthetize flies</td><td>Tritech Research</td><td>MINJ-DROS-FP</td><td></td></tr><tr><td>1 L cage</td><td>Web Restaurant Store</td><td>999RD32</td><td></td></tr><tr><td>1 L cage lid</td><td>Web Restaurant Store</td><td>999LRD</td><td></td></tr><tr><td>a glass-teflon dounce homogenizer&amp;nbsp;</td><td>Fisher Scientific</td><td>NC9661231</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>rapamycin&amp;nbsp;</td><td>LC Laboratories&amp;nbsp;</td><td>R-5000</td><td></td></tr><tr><td>anti-porin</td><td>MitoSciences</td><td>MSA03</td><td></td></tr><tr><td>anti-alpha tubulin</td><td>Developmental Studies Hybridoma Bank</td><td>12G10</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>23225</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pad</td><td>Tritech Research, Inc</td><td>MINJ-DROS-FP</td><td></td></tr><tr><td>filter flask</td><td>enasco</td><td>SB08184M</td><td></td></tr><tr><td>rubber stopper</td><td>enasco</td><td>S08512M</td><td></td></tr></tbody></table>

preparation of mitochondrial enriched fractions for metabolic analysis in drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

使用的协议如上所述，我们进行了对线粒体富集级分和整个动物提取物的代谢组分析测试药物雷帕霉素对发散线粒体DNA 7的效果。我们通过在飞食品溶解药物递送200微米的雷帕霉素。蝇暴露于雷帕霉素10天。从整体蝇提取物和从线粒体提取物代谢物通过采用气相色谱质谱（GC / MS）和液相色谱 - 串联质谱（LC / MS / MS），使用标准的溶剂萃取方法获得。 图1显示了膜转运蛋白孔蛋白线粒体富集在线粒体级分指示器的富集。孔蛋白是检测不到的胞质部分。相反，在线粒体级分，苏没有检测到微管蛋白ggesting胞质蛋白的小的污染。从"本地"D。 图2和图3示出主要部件的完整代谢物轮廓分析（PCA） 果蝇株（ORER）和苍蝇D.窝藏线粒体DNA的&#39;破坏&#39;应变simulans和四核DNA 果蝇应变ORER（SM21）。两种菌株暴露于药物雷帕霉素。数据被呈现为双情节主成分的轴1和2来可视化处理和线粒体DNA 7,9的效果。使用标准的整个飞行提取物中获得的代谢产物示于图2A中。图2B示出了类似的曲线图的PCA在线粒体富集提取物的代谢物。对于整个蝇提取物（图2A），雷帕霉素治疗进行筛选样本以PC2的显著较低值。然而，由于线粒体DNA创变化的代谢产物otype是微妙的，因为暴露于雷帕霉素或控制车辆ORER和SM21线粒体DNA是紧密相邻的或重叠的PCA空间。然而，使用富集的线粒体提取物时，线粒体DNA显示对线粒体代谢物轮廓分明的雷帕霉素的效果。特别是，雷帕霉素对ORER菌株的代谢物轮廓，通过沿PC1轴线的位移显示出强大的影响。但是，在"中断"SM21线粒体DNA基因型，对于控制食品的条件代谢物图谱是从天然ORER应变的移位（红色和黑色的多边形，分别在图2B中）。其结果是，雷帕霉素治疗具有明显较少的对代谢物的景观中被破坏的线粒体DNA基因型，SM21移位（比较红和蓝的多边形在图2B中）的效果。 图3示出的PCA p许多参与能量稳态代谢物（所有代谢的子集，限于辅因子，维生素和三羧酸循环中间体），常位于线粒体内。这里再一次，从富集的线粒体级分的代谢物转移显示出比那些从整个动物提取物不同的行为。 这些结果说明通过线粒体富集提取物和整个动物提取物中获得的信息如何代谢不同，但相互补充，与线粒体富集提取物是在线粒体代谢偏移更敏感。 <img alt="图1" src="/files/ftp_upload/53149/53149fig1.jpg" /> 图1:Western blot分析显示线粒体在线粒体分数有效分离。使用的抗体反对孔蛋白（一种线粒体外膜蛋白）和微管蛋白（胞浆蛋白）。蛋白质<html>丰度的线粒体和细胞质级分用二辛可宁酸（BCA）测定法进行定量。总蛋白的30微克加载在每个泳道。两个独立的提取进行。 <img alt="图2" src="/files/ftp_upload/53149/53149fig2.jpg" /> 采用整个动物的样品与对苍蝇携带ORER线粒体富集提取物和SM21线粒体单倍型获得代谢物如图2主成分分析（PCA）。的整个动物样品中鉴定的代谢物210（A）中的PCA。 （B）PCA对线粒体提取物检测到230代谢产物。周围点的面，目的是帮助六个复制样本的可视化对于每个治疗。K"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53149/53149fig3.jpg" /> 参与能量平衡的苍蝇携带ORER和SM21线粒体单倍型的代谢产物图3.主成分分析。从整个飞提取物12的能量代谢和线粒体提取物13的能量代谢产物（A）PCA。 （二）围绕点的面，目的是帮助六个复制样本，每个处理的可视化。 <a href="https://www.jove.com/files/ftp_upload/53149/53149fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila at JoVE.com

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

preparation-mitochondrial-enriched-fractions-for-metabolic-analysis

Universidad Científica del Sur

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K 次观看.  Adelphi University. Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster. 

视频: Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

用最小组织量测定果蝇透纤维中线粒体耗氧量

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

科研

生物化学

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

发育生物学

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

生物学

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

<meta charset="utf8"></head><body>一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

行为学

分析果蝇的线粒体生理学和生物能量学

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

作者聚焦：使用黑腹果蝇探索线粒体功能和化学毒性 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

在代谢分析准备线粒体富集级的

摘要

探索更多视频

用最小组织量测定果蝇透纤维中线粒体耗氧量

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

从酵母细胞线粒体的纯化

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

果蝇幼虫和成年脑的代谢分析

在代谢分析准备线粒体富集级的

摘要

探索更多视频

用最小组织量测定果蝇透纤维中线粒体耗氧量

果蝇梅拉诺加斯特的锡酸盐合成酶活性的色度测定

基于气相色谱-质谱 (gc-ms) 的新陈代谢的果蝇幼虫样品的制备

从酵母细胞线粒体的纯化

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果 蝇 行为研究平台，以及幼虫线粒体呼吸测定的配套方案

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析 果蝇黑腹 果蝇 PINK1B9-Null 突变体的线粒体功能

果蝇幼虫和成年脑的代谢分析

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

一个经济实惠且高效的“自制”果蝇行为研究平台，以及幼虫线粒体呼吸测定的配套方案

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能

使用高分辨率呼吸测量法分析果蝇黑腹果蝇 PINK1^B9-Null 突变体的线粒体功能