The author thanks Drs. Mingjian Lu and Morris Birnbaum for helpful advice and funding support from NIDDK (R21DK089391).

1.飞食品制备含有放射性标记代谢底物<ol><li>在离心管中，混合1 - 2微居里放射性标记的底物（D- [6- 14 C]葡萄糖，D- [1- 14 C]葡萄糖或[1- 14 C] -棕榈酸，40 - 60居里/毫摩尔）与1月15日微升FD＆C号蓝色食用染料和H 2 O等于​​25微升的总体积。 注意:碳14是一种低能量β发射体，并具有在空气中很短的范围内。但是，要小心防止放射性标记分子或实验者意外摄取的溢出。飞食品瓶翻倒容易;一定要容纳它们的容器中，这将防止其翻倒，特别是当放射性标记是液体形式（步骤1.2），或在未凝固食品（步骤1.4）。正在馈送或已被馈送放射标记底物将开始呼气少量的放射性标记的 CO 2的苍蝇。这些苍蝇应保存在亚克力箱在通风柜，和一小碟KOH的CaN为在用作CO 2洗涤器的框设置。 </li><li>吸管所有放射性标记的底物和蓝色染料混合到一个空的飞食品小瓶的底部（高度:9.5厘米，宽2.2厘米）。 </li><li>热标准飞食物在微波炉中，直至食物被刚液化（15 - 在功率电平高于含有10ml食品之一药瓶20秒）。 </li><li>加入975微升熔融食品放射性标记的基板/蓝染料混合，迅速漩涡，监测蓝色的均匀性，以确保完全混合。 </li><li>允许食物冷却，并在室温完全固化20 - 30分钟。 </li></ol> 2.饲喂放射性标记代谢底物，以成人果蝇<ol><li>麻醉成年使用由稠合到丙烯酸基和连接到与压力调节器的 CO 2罐多孔聚乙烯的在 CO 2麻醉装置苍蝇。以5升/分钟流动的 CO 2进入麻醉装置。 <ol><li>麻醉转移ð飞往含有放射性标记，蓝染食品瓶（N = 15 - 每瓶30苍蝇）。盖小瓶泡沫塞，水平休息，直到苍蝇醒过来。转移小瓶带盖容器压克力（180厘米×180厘米×240厘米高）。 </li></ol></li><li>转移苍蝇放射性标记的食物2前24小时- -短期饲养，饿死18苍蝇。3小时供给工序吨他饥饿步骤非常重要，因为美联储苍蝇就不能可靠地吃呈现给他们食物的新来源。对于长期馈送超过5的过程中 - 第7天，苍蝇不需要饿死，但实验者应监测放射性标记的食品在苍蝇都装在小瓶中的水含量，并使用针和注射器，以水添加到小瓶带干粮。 </li><li>通过转移"窃听"他们到一个新的小瓶飞往未标记的食物。 2最初的追逐时期 - 4小时允许苍蝇从他们的角质层清除放射性标记的食物颗粒，并允许放射性标记的消化ED剩余的食物在肠道。显示器通过苍蝇的视觉检查肠道的食物进度寻找蓝色腹部。 </li><li>随后，转飞到不同的食品种类（12 - 标准的食品或1％，琼脂24小时的的影响测量与喂养对呼吸空腹状态）或环境温度（12 - 96小时在18℃或30℃遗传使用温度敏感GAL80（GAL80 TS），对例如操作）。 注意:此追期间的长度将与正在执行的实验而变化。为例如 ，在30℃下的96小时追逐可能有必要在使用GAL80 TS为了实验充分激活GAL4依赖性转基因表达。 </li></ol> 3. 二氧化碳 -Collection装置的制备<ol><li>组装将包含标有14的C-葡萄糖或14 C-棕榈酸和Tha苍蝇材料建造"飞行舱"，网皑皑管ŧ将继续开放到大气中。切断为12毫米×75毫米的聚丙烯管的前50毫米，留下25毫米长，圆底管。准备130微米的尼龙网35毫米×35平方毫米。此外，获得的胶带分配器透明胶带。 </li><li>集合， 二氧化碳 -collection设备材料。对于每个飞荚，组装20毫升玻璃闪烁瓶中，橡胶顶端止动件与偏心孔，以及通过孔在顶部塞子被插入一个中心，级GF / B玻璃微纤维滤纸（2.1厘米直径的圈，1微米孔径）。 GF / B滤纸的使用，因为它的高的湿强度和高承载能力。 </li><li>上使用的当天制备5％的KOH的新鲜。只是步骤3.4之前，折叠和圆形GF / B滤纸放入中心孔即通过橡胶顶端止动件的孔螺纹并用100μl5％的KOH的饱和它。参见图1。 </li><li>继未标记的媒体，一个孵化（S）nesthetize苍蝇与二氧化碳 。刷机飞入截止聚丙烯管，顶盖采用尼龙网，并用透明胶带坚持网格管。迅速开展工作，以避免苍蝇醒来，并在逃跑前网已经牢固地安装在管。苍蝇的数量不变应在每个飞荚特定实验中使用; 10 -每荚飞蝇20产生足够的放射性-CO 2通过闪烁计数（下面的步骤4.1）测量。 </li><li>传送飞吊舱20毫升玻璃闪烁瓶中。帽与橡胶顶端止动件保持孔含有KOH饱和GF / B滤纸的中央的玻璃小瓶。折叠橡胶顶端止动件的宽顶部在玻璃闪烁小瓶的唇。参见图1。 </li><li>包含在一个带盖，丙烯酸容器蝇集玻璃小瓶中，孵育的时间（2〜12小时工作良好）不同时期。 </li></ol> 4.结果分析<ol><li>在温育后，摘帽玻璃小瓶中，转移的KOH饱和GF / B滤纸上含有4毫升闪烁鸡尾酒6毫升的塑料闪烁小瓶的端部。 </li><li>如果需要，冻结干冰，并存储在飞荚苍蝇在-80℃存储基板后确定。请注意，这些样品是放射性的，并且必须相应地存储。 </li><li>另外准备闪烁瓶:含有未GF / B滤纸作为背景之一，含0.1差一分二点别人 - 0.5微居里放射性基板，以确定CPM /微居里。 </li><li>测量使用根据制造商的方案用闪烁计数器每个样品的cpm。 </li><li>计算皮摩尔14 CO 2 / CPM和皮摩尔14 CO 2的每飞行每小时。减去每个样品的CPM值背景CPM。从整齐的放射性基板的数据，计算出背景校正CPM /样品微居里计数。 <ol><li>使用制造商提供的特定活性和放射性标记的底物的放射化学浓度（例如，50微居里/微摩尔和0.1微居里/微升，分别）来计算皮摩尔/ CPM。使用此因素的背景校正飞行荚数据CPM 14 CO 2 /采样转换为皮摩尔14 CO 2 /样品。然后通过昆虫的数量和hr的在飞荚数分裂。 </li></ol></li></ol>

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The author declares no competing financial interests.

这个协议描述了测量在成年果蝇特定放射性标记底物的氧化的方法。这种技术是直接的，定量的，可再现的和敏感的，并且它补充了测量总的 CO 2生产和O 2消耗，因为它可被用来识别被氧化为ATP产生的养分（多个）的现有方法。 使用这里描述的方法的特定的放射性标记底物的氧化释放的CO 2的测量是该测量总的 CO 2的生产技术是互补的。总的 CO 2生产（ⅤCO 2）允许代谢率的估计，并且当总的 O 2消耗（V 02）是已知的，代谢率可以计算和用于氧化燃料源可以基于所述呼吸交换率估计（Ⅴ CO2 / V O2 = RER）。什么时候碳水化合物是独占的衬底，RER = 1，但是，当脂肪酸是异源，RER = 0.7，由于葡萄糖和脂质氧化的反应的化学计量。在没有设备来测量在果蝇 14氧消耗，氧化燃料源不能被识别。然而，通过标记与一种放射性底物或另一种痕量和放射性标记的CO 2的生产的测量速率苍蝇，人们可以得出关于苍蝇的刺激过程中或约的效果以氧化在不同时间点的给定基底的能力的结论鉴于燃油氧化突变。 有许多在该协议的关键步骤。首先，应注意使用放射性物质，以避免泄漏和研究领域的污染作出。二，麻醉过程中的步骤2.1和3.4苍蝇时，实验者应迅速工作，以避免长时间的 CO 2的效果曝光的代谢和行为。 A组20苍蝇可麻醉并转移至新的小瓶或到20秒或更小的飞吊舱。当从麻醉设备转移到食物小瓶中，小瓶应当在其一侧被休息以防止麻醉蝇从卡住对食品的表面上。当苍蝇在数天的过程中馈放射性标记食品如在步骤2.2中，实验者应确保该食品不干透苍蝇是脱水敏感。 进食或禁食状态之前标签，标签的选择，标记的时间长度，时间在飞荚长度，和麻醉的方法是可以使用这种技术时，可以进行故障诊断和修正参数。首先，苍蝇经过短标签周期（2 - 3小时）不太可能，除非经受禁食期间提前消费的食品放射量显著。第二，标记C的选择有影响的结论之一就是能够得出代谢表型。例如，从D-放射性标记的CO 2的生产[1- 14 C]葡萄糖能反映通过柠檬酸循环或戊糖磷酸分流氧化，而 ​​从D-放射性标记的 CO 2的生产[6- 14 C]葡萄糖反映氧化通过三羧酸循环只有12,15,16。馈送带放射性标记的示踪物的苍蝇为一种必需氨基酸17,18将是评估从蛋白质衍生的氨基酸氧化的好策略。最后，用放射性标记的葡萄糖冗长标记周期也提高到其它类分子，如甘油三酯19和氨基酸，如丙氨酸，易于与丙酮酸interconverts该前体中掺入的可能性。这可通过测量放射性掺入这些不同类分子6的进行评估。事实上，苍蝇独立的同伙可以喂拉德以相同的方式iolabeled基底，然后用于存储和在馈保持在糖原和甘油三酯和禁食条件6，例如苍蝇荚果实验或放射性标记的测量。另一个策略是使用有可能揭示本身放射性标记的膳食的营养成分的氧化和这些营养素不存储形式的短标记周期。第三，它也有可能是蝇两组可具有14 CO 2的产量超过给定的时间量的相同速率，但很不同的速率最初。因此，不同的时间是拥有在飞荚花费的金额可以是评估表型变异时改变的重要参数。最后，该协议要求使用二氧化碳麻醉苍蝇把成飞荚设备时短时间的。这是可能的CO 2的麻醉可能影响代谢功能在飞荚实验过程。另一种一接近角是使用氮麻醉不影响代谢速率20。 与测量这样一个复杂的系统，燃料的氧化和代谢速率的任何技术，这里所描述的方法具有局限性。首先，它可能是在不同的组可以消耗不同量的放射性标记的食品苍蝇，这将与衬底的不同起始量进入该测定的CO 2的收集阶段。这可以通过执行与大样本实验和饲养苍蝇成群加以控制在一定程度。短标签时期，过得有类似蓝色的胆量都将消耗的放射性标记，可以结转到实验的追逐部分。剩余的一组蝇中的放射性标记的量也可以在实验结束时，并在进料和初始追逐期间结束后的苍蝇单独队列测量。第二，之间的活动水平在飞豆荚苍蝇基团可以不同。这将必须通过实验来确定，并解释数据时加以考虑。第三，该技术不测量总CO 2的生产，只是从一个特定的食物营养或营养的存储形式（多个）的生产。这个协议是不进行更换，而是对VCO 2的测量互补，因为它提供了不同的和额外的信息。 这里所描述的方法测量呼出，放射性标记的 CO 2是特定的代谢底物的痕量的氧化的产物。通过改变所用的标记 - 葡萄糖，脂肪酸，或氨基酸 - 可以设计越来越复杂的实验，以评估不同的基材的代谢不同的生理条件如饥饿和在不同的遗传背景下做出的贡献。这种技术的未来应用包括metabolis测量米发展的幼虫和蛹的阶段， 果蝇的生命周期为特征的营养分别储存和分解，极端的两个阶段。

在模式生物果蝇的工作已经到遗传原理，发展过程，成长，衰老，行为，免疫和人类疾病1,2的理解作出了巨大贡献。在果蝇遗传学和细胞生物学方法一无数在这些领域带动发展。然而，新陈代谢在果蝇的研究已经发展较为缓慢，在很大程度上是由于在测量这样的小动物代谢参数的困难。在使用果蝇作为研究人类疾病如糖尿病的模型和对于理解代谢的增长和各种病症的捐款的兴趣已经被推领域开发和适应代谢技术这种微生物3,4。 可靠的方法现已为多家在果蝇代谢参数的测量。例如，它是简单的评估食物摄取<s向上&gt; 5，存储甘油三酯和糖原，以及葡萄糖的循环血淋巴水平和在飞，海藻糖，这是一个葡萄糖4的两分子组成的双糖的主要循环糖水平。代谢示踪剂的使用使从饮食和吸收的营养的转化营养吸收的研究如葡萄糖到存储形式的糖原和甘油三酯6。代谢率可以在水果通过氧消 ​​耗7,8和二氧化碳的测量飞行（CO 2）的生产来评估。三羧酸循环氧化二碳单位，可以进入循环为乙酰辅酶A（辅酶A），它是从饮食和存储碳水化合物和脂肪酸的代谢而得。该循环的每个依次产生每个GTP（或ATP）的一个分子和电子给体FADH 2，NADH的三分子，和废产物的 CO 2的两个分子。 CO 2的产能被用于估计的基础代谢率。总的二氧化碳生产可以在果蝇通过使用市售的或手工呼吸计9-10,11的量化。 总的 CO 2生产的测定中，特别是当与O 2的消耗测量耦合，提供了有价值的洞察全身能量代谢。然而，这一措施不标识养分被氧化为三磷酸腺苷（ATP）的生产。营养素的三大类可以进入三羧酸循环以下转化为乙酰辅酶A:碳水化合物，脂肪酸，和蛋白。葡萄糖-6-磷酸，从膳食葡萄糖或储存的糖原衍生，可以转换到由丙酮酸脱氢酶脱羧以形成乙酰CoA丙酮酸盐。通过β-氧化收率乙酰CoA该再进入三羧酸循环从饮食或从存储的甘油三酯的脂肪酸击穿。最后上，大多数氨基酸可进入三羧酸循环以下转化为丙酮酸，乙酰辅酶A或三羧酸循环的中间产物如α-酮戊二酸。 基底和刺激条件期间能量代谢特定营养贡献可以通过使用放射性示踪剂的进行监测。这里介绍的协议适于在果蝇使用从用于评估在培养12,13生长的细胞氧化的协议。在这种方法中，果蝇是在饮食很短的时间（小时）馈14 C-放射性标记的代谢底物（碳水化合物，脂肪酸，或氨基酸）或长（天）脉冲，追赶上未标记食品，然后暴露于包含氢氧化钾（KOH） -饱和滤纸用于捕集呼出的CO 2的碳酸氢钠室中。放射性标记的碳酸氢盐可以通过闪烁计数进行定量。在实验的放射性-CO 2的生产差异苍蝇的人团可以反映使用不同的燃料的对ATP产量超过在燃料代谢基因型之间延长的快速或固有的差别的过程中，例如。

<table><tbody><tr><td>PALMITIC ACID, [1-14C]-, 50 &amp;micro;Ci</td><td>PerkinElmer</td><td>NEC075H050UC</td><td></td></tr><tr><td>GLUCOSE, D-[6-14C]-, 50 &amp;micro;Ci</td><td>PerkinElmer</td><td>NEC045X050UC</td><td></td></tr><tr><td>Glucose, D-[1-14C]-, 50&amp;micro;Ci</td><td>PerkinElmer</td><td>NEC043X050UC</td><td></td></tr><tr><td>Drosophila Vials, Narrow, Polystyrene</td><td>Genesee Scientific</td><td>32-116</td><td></td></tr><tr><td>Round-Bottom Polypropylene Tubes, 12 x 75 mm</td><td>Fisher Scientific</td><td>14-959AA</td><td></td></tr><tr><td>Mesh, nitex nylon, 120 &amp;micro;m</td><td>Genesee Scientific</td><td>57-102</td><td></td></tr><tr><td>20 ml borosilicate glass scintillation vials</td><td>Fisher Scientific</td><td>03-337-15</td><td></td></tr><tr><td>Flask top stopper with off-center hole</td><td>Fisher Scientific</td><td>K882310-0000</td><td></td></tr><tr><td>Polypropylene center well</td><td>Fisher Scientific</td><td>K882320-0000</td><td></td></tr><tr><td>Whatman GF/B glass microfiber filter paper, circle, 2.1cm diameter</td><td>Fisher Scientific</td><td>09-874-20</td><td></td></tr><tr><td>Ecoscint A</td><td>National Diagnostics</td><td>LS-273</td><td></td></tr><tr><td>6 ml Scintillation Vials with Push-On/Twist-Off Caps</td><td>National Diagnostics</td><td>SVC-06</td><td></td></tr></tbody></table>

measurement of carbon dioxide production from radiolabeled substrates in drosophila melanogaster

果蝇遗传学的电源被越来越多地应用到激素信号和代谢的问题，并在对人类疾病模型在该生物体的发展。需要如代谢率的参数测量敏感的方法，以推动小动物生理和疾病的认识，例如果蝇。此处所描述的方法评估的燃料氧化饲喂含14 C标记的底物如葡萄糖或脂肪酸痕量食品成蝇小的数字。馈送周期以及任何其他实验操作后，蝇转移至网眼封短管，然后将其放置在含有KOH饱和滤纸的玻璃小瓶该陷阱呼出，从放射性标记的底物如碳酸氢钾的氧化产生的放射性标记的 CO 2， KHCO 3。此放射性标记的碳酸氢盐是通过闪烁计数来测量。这是AQ燃料氧化研究uantitative，重复性好，简单的方法。使用放射性标记的葡萄糖，脂肪酸，或氨基酸的允许这些不同的燃料源到能量代谢不同的条件，如进料和禁食和在不同的遗传背景下作出的贡献的判定。这补充用于体内能量代谢以测量其他方法和应进一步代谢调节的理解。

呼出和截留的放射性标记的 CO 2是从放射性底物代谢中产生的量应与苍蝇在苍蝇荚数量以及时间的动物中的CO 2的收集装置花费的金额相关联。为了测试这一点，禁食18小时，即蝇饲喂放射性标记的棕榈酸（0.02微居里/微升食品）2小时，然后用于放射标记的CO 2的生产在12或25只动物的组进行3或6小时的孵育测试。 的 CO 2的25个蝇产生的量几乎加倍12苍蝇产生的量，并为每个组的数量增加了一倍，当孵育时间从3小时提高到6小时（ 图2A）。在皮摩尔14 的 CO 2 /飞/小时，各组中几乎相等。 空腹由ßØ刺激脂肪酸的分解xidation;因此从脂肪酸氧化的 CO 2生产中应在禁食动物可升高与馈动物相比。为了确定这是否是的情况下，18小时禁食蝇饲喂放射性标记的棕榈酸2小时。以下这个短馈送脉冲，苍蝇分为两组，并转移到未标记的飞食品或1％琼脂3小时，然后转移到飞吊舱2小时。在两个独立的实验中，苍蝇追赶琼脂上产生显著更放射性标记的 CO 2与追赶上的食物（图2B）蝇相比，表明SS氧化反应在禁食动物增加。 <img alt="图1" src="/files/ftp_upload/54045/54045fig1.jpg" /> 图1. 该装置用于收集呼出，放射性标记的 CO 2。 过去那种消耗放射性代谢底物的苍蝇放入"飞荚"（FP），截止离心管目（M）封端。飞荚放入20ml的玻璃闪烁小瓶中并加盖用含有偏心孔包含用100μl饱和GF / B滤纸的橡胶顶塞（S），通过它被置于一个中心孔（CW） 5％的KOH。呼出的 CO 2与氢氧化钾反应，被捕获在滤纸上作为碳酸氢盐。困，放射性标记的二氧化碳可以通过饱和滤纸闪烁计数定量。 <a href="https://www.jove.com/files/ftp_upload/54045/54045fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54045/54045fig2.jpg" /> 图 2. 时间 14 CO 2 生产相关，蝇在数飞波德与应对美联储和禁食条件。 （A）在果蝇产生的14 CO 2的供给放射性棕榈酸的量与测试苍蝇的数目有关（N = 12（空心柱）或25（闭合条））和时间在苍蝇荚花（3或6小时）。在每个飞基础，苍蝇产生0.99，1.13，1.10和1.01皮摩尔14 的 CO 2 /小时（在相同的左列向右顺序作为图形数据的数据）。 （二）被追赶上的琼脂氧化更多放射性比棕榈追了关于食物，苍蝇苍蝇。数据来自两个实验和报告为装置标准偏差，N = 16 - 17蝇/组（实验1）;每组10只苍蝇/组（实验2）。 * P = 0.017，学生t检验。 <a href="https://www.jove.com/files/ftp_upload/54045/54045fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster at JoVE.com

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 ...

measurement-carbon-dioxide-production-from-radiolabeled-substrates

Research

JoVE Journal

Biology

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

9.1K 次观看.  University of Virginia. 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.

视频: Measurement of Carbon Dioxide Production from Radiolabeled Substrates 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

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 ...

生物化学

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 ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

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 ...

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 ...

神经科学

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 ...

适用于 果蝇 的微量呼吸测定法 - 深入了解 CASK 突变体代谢

Measuring O2 Consumption in Drosophila melanogaster Using Coulometric Microrespirometry

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable environment. A coulometric microrespirometer consists of an airtight chamber in which O2 is consumed and the CO2 produced by the organism is removed by an absorbent medium. The resulting pressure decrease triggers electrolytic O2 production, and the amount of O2 produced is measured by recording the amount of charge used to generate it. In the present study, the method has been adapted to Drosophila melanogaster tested in small groups, with the sensitivity of the apparatus and the environmental conditions optimized for high stability. The amount of O2 consumed by wildtype flies in this apparatus is consistent with that measured by previous studies. Mass-specific O2 consumption by CASK mutants, which are smaller and known to be less active, was not different from congenic controls. However, the small size of CASK mutants resulted in a significant reduction in O2 consumption on a per-fly basis. Therefore, the microrespirometer is capable of measuring O2 consumption in D. melanogaster, can distinguish modest differences between genotypes, and adds a versatile tool for measuring metabolic rates.

作者聚焦:温度对黑腹果蝇和沙漠适应性甲虫的影响 

Coulometric respirometry is ideal for measuring the metabolic rate of small organisms. When adapted for Drosophila melanogaster in the present study, measured O2 consumption was within the range reported for wildtype D. melanogaster by previous studies. Per-fly O2 consumption by CASK mutants, which are smaller and less active, was significantly lower than the wildtype.

使用库仑显微呼吸法测量黑腹果蝇的 O2 消耗量

Coulometric microrespirometry is a straightforward, inexpensive method for measuring the O2 consumption of small organisms while maintaining a stable ...

从放射性标记的底物在二氧化碳生成的测量

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