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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	<li>Scheffler, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondria (Scheffler, Mitochondria). , 484 (2007).</li><li>Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria--a+nexus+for+aging,+calorie+restriction,+and+sirtuins.">Mitochondria--a nexus for aging, calorie restriction, and sirtuins.</a> Cell. 132 (2), 171-176 (2008).</li><li>Raimundo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathology:+stress+signals+from+the+energy+factory.">Mitochondrial pathology: stress signals from the energy factory.</a> Trends in molecular medicine. 20 (5), 282-292 (2014).</li><li>Embley, T. 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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...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.2% tegosept -methyl 4-hydroxybenzoate&#160;</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-(N-morpholino) propanesulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td>M1254&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>38057&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T-B7</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551139</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 pads to anesthetize flies</td>
			<td>Tritech Research</td>
			<td>MINJ-DROS-FP</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999RD32</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage lid&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999LRD</td>
			<td></td>
		</tr>
		<tr>
			<td>a glass-teflon dounce homogenizer&#160;</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&#160;</td>
			<td>LC Laboratories&#160;</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&#8482; BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 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。 <p class="j...

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 ...

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Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.4K 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. 