The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

注:该协议遵循亚利桑那州办公室的大学研究的负责行为准则和已批准的机构动物护理和使用委员会 1.斑马鱼繁殖和胚胎维护<ol><li>准备E3胚胎中期8。为了使60X贮备液，溶解34.8克氯化钠1.6克氯化钾，氯化钙5.8克2●2H 2 O和9.78克氯化镁 ●6H 2 O在1.95大号ddiH 2 O.调节pH至7.2用NaOH和HCl调节音量用ddiH 2 O 2升</li><li>为了准备1X E3胚胎中期，淡化16.5毫升60倍的股票在1升ddiH20。加入100微升1％亚甲蓝。 </li><li>选育<ol><li>宅男和女亲鱼（7 - 18个月）分别以最大限度地提高繁殖力。 </li><li>前2小时，当天鸡蛋所需的前 - 关灯1。建立交配（1男1 - 2名女性）在C罗辛笼如前所述9。渡网箱允许鸡蛋逃脱捕食，因为他们通过在成鱼保持油箱插入的穿孔基地通过。 </li><li>择:对于定时育种，（如，将需要涉及吗啉注射研究），分离雌雄鱼由透明塑料分频器，在上午除去此导致卵受精的定时铺设。对于吗啉的研究，进行功率计算，以评估每次治疗所需的胚胎的鱼的数量。没有效果或变化的幅度的估计，一个初步试验可有n = 8运行。 </li></ol></li><li>倾从交叉笼将鸡蛋打入培养皿（100 - 150胚胎/ 10厘米培养皿）。允许卵沉淀到培养皿的底部，并倒出水。用细尖一次性移液管中取出所有剩余的水。加回步骤1.1取得E3胚胎中期。 </li><li>使自动对焦跛脚抛光一次性吸管为今后的蛋和鱼转移。用剪刀剪毕业一次性移液管以0.25ml毕业。要删除粗糙的边缘，火焰抛光切割端过本生灯。 </li><li>在斑马鱼胚胎的E3介质的培养皿在28.5○C.保持胚胎鱼至4天受精后（dpf）的10厘米每天两次，用火焰打磨毕业一次性移液管，以消除任何未受精的卵子或胚胎死亡（不透明的白色外观）。每天一次，更换E3胚胎中期。倒掉E3胚胎培养基，用细尖一次性移液管除去残留的液体，加回预热（28.5○C）E3胚胎中期。 </li></ol> 2.准备分析解决方案<ol><li>根据使用0.22微米过滤器表1.无菌过滤器测定溶液制备测定溶液。在测定前启动，暖在28.5○C无菌检测解决方案水浴。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td>成分</td><td> ％的总</td><td>卷（微升/ 10毫升） </td></tr><tr><td> 60X E3胚胎中期</td><td> 1.667 </td><td> 166.7 </td></tr><tr><td>双去离子（DDI）H 2 O </td><td> 96.233 </td><td> 9623.3 </td></tr><tr><td>阿尔玛蓝</td><td> 1 </td><td> 100 </td></tr><tr><td> 400毫米氢氧化钠</td><td> 1 </td><td> 100 </td></tr><tr><td> DMSO </td><td> 0.1 </td><td> 10 </td></tr></tbody></table> 表1.分析培养基 3.执行分析<ol><li>冲洗斑马鱼胚胎3次，无菌过滤E3胚胎中期。将所有可行的斑马鱼胚胎从离合器放入烧杯中。无菌过滤器75毫升E3胚胎中期。用细尖一次性移液管，重新从斑马鱼胚胎尽可能将尽可能多的E3胚胎中期的。添加倒退20毫升无菌过滤E3胚胎中期。卸下并更换E3胚胎中期的3倍。 </li><li>板1成鱼每93孔96孔黑色的双面明确的底板。通过单独捕获胚胎内的火焰磨光毕业一次性塑料移液管（步骤1.5），轻轻地喷射到井内的胚胎鱼转移到板上。从转移含有清洗鱼放入3空白孔烧杯中采取E3胚胎中期。这确保了空白孔被暴露了完全相同的水作为含有鱼类的孔中。包含鱼和空白孔两口井应至少半满（200微升）在这一步的结论。 </li><li>更换E3胚胎中期，在所有96个孔，其中包括空白孔检测解决方案。此步骤，应执行1列的时间为每个在96孔板的12个列。使用精尖的一次性反FER吸管，从每一个井板（8孔）的一列去除E3胚胎中期。小心不要碰斑马鱼胚胎。添加300微升测定溶液至每孔从其中除去水。重复使用的板的所有12列测定溶液去除E3胚胎中期和更换。 </li><li>择:为了测试响应于化合物的代谢速度的变化，使一个解决方案，是100倍（100X）所需的治疗浓度。值得注意的是，确保了100倍的溶液不超过10％的DMSO，以限制最终DMSO浓度到不超过0.1％以上。加入3微升100倍的溶液，以各处理井。 注:执行功率计算以评估治疗井的数量。然而，如果没有响应或变化的大小的估计，一个初步试验可在处理8化合物和包含鱼8载体治疗的对照孔中运行。 <ol><li>加入3微升车辆控制来控制（没有DRU摹处理），包含鱼井。为了确保到化合物的荧光响应是的鱼中的动作的结果，应用药物和车辆处理，以3空白孔在96孔板中。 </li></ol></li><li>阅读荧光酶标仪激发波长为530 nm，发射波长为590纳米的鱼填板。 </li><li>将板进入加湿孵化器设置为28.5○C（保持板在黑暗整个试验，以避免测定溶液漂白）。该分析方法可用于持续时间的范围从1小时至24小时运行。测定法的持续时间取决于研究的目标。用于测试响应于短效非稳定化合物短的持续时间可能是最适合的。然而，对于稳定的化合物测试或遗传效应试验，最大限度地提高检测的持续时间最好利用与累积的信号相关的优点。 </li><li>在预先确定的时间点（见步骤3.6，以帮助确定时序）使用相同的设置步骤3.5再读鱼填充板的荧光。 </li></ol> 4.评估在荧光相关的相对变化与治疗<ol><li>井通过从荧光减去荧光在时间0在研究结束时计算变化中的每个的荧光。井无鱼（空格）应包含鱼井还有一些值接近0，远低于价值。 更改荧光=荧光的结论 - 荧光在时间0 </li><li>为了确定在荧光的相对变化，计算在荧光的平均变化对于对照孔，然后通过在对照孔的荧光的平均变化除以在的各孔的荧光变化。值得注意的是，对照可以是载体对照或基因控制。 <img alt="式（1）" src="/files/ftp_upload/53297/53297eq1.jpg" /></li></ol>

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The authors have no competing financial interests to disclose.

污染与细菌或真菌将极大地限制了应用此法的。在E3胚胎培养基中的亚甲基蓝限制真菌污染的可能性。在整个胚胎饲养至关重要的是，小心一天两次，每次去除死胚胎。此外，有必要彻底冲洗用无菌的E3胚胎培养基中的胚上测定引发的天。这些2步限制用于胚胎细菌污染的可能性。列入步骤3.2中所述空白孔中可以检测任何细菌污染。细菌污染而产生可测量的信号是罕见的。然而，如果空白孔做显示出强大的信号，所述测定应重复小心以保持胚胎清洁度服用。如果用户期望污染前胚胎收集所产生的，如先前所述（第22页）10中的胚胎可以是漂白。或者，如果实验者怀疑污染物收集后发生的用户可以使用E2与青霉素和链霉素到后的胚胎4 DPF（第23页）10。 虽然这个实验是在胚胎的应用价值，我们还没有在成年斑马鱼的有效性进行测试。我们怀疑这个实验的功能将在成鱼用的AlamarBlue的穷人组织渗透是有限的。此外，食性鱼类肠道菌群可能会产生深远的信号，并不表明鱼的新陈代谢。因此，该测定被限制在蛋黄喂养鱼胚胎使用。胚胎鱼类的相对不活动提供了一个独特的优势，因为这限制了将与活动引起的能量消耗11相关联的可变性。因此该测定是一个独特的工具来筛选在整个动物系统中的代谢率响应于遗传或药理学处理。 斑马鱼正迅速成为并购的主要模式etabolic扰动伴随肥胖。事实上，有公布既转基因和饮食诱导的肥胖斑马鱼12,13的型号。类似的小鼠模型，在斑马鱼的饮食引起的肥胖增加血清甘油三酯和肝脂质蓄积12。此外，吃得太多斑马鱼已空腹血糖升高，提示葡萄糖不耐受14。使用此处描述我们已经表明，预处理二甲双胍，胰岛素增敏剂，24小时增加代谢率响应于胰岛素治疗5测定。因此，斑马鱼可能被证明是一种理想的整个动物模型来发现的药物，增强胰岛素敏感性（胚胎），并建立它们的效力，以减轻肥胖相关的胰岛素抵抗（成人）。 模仿肥胖哺乳动物中观察到的表型的遗传和饮食诱导的肥胖斑马鱼模型的发展创造了动力启动斑马鱼STU模具专注于代谢性疾病。高通量测定是为充分理解斑马鱼模型的值，以能量稳态研究必不可少的。旨在评估phagic驱动创新工具将允许屏幕集中在能量摄入，而高吞吐量脂质贮积试验将导致对整个身体脂质平衡15-17研究。我们在这里描述的测定法提供了一种高通量工具，以评估基因，化合物或它们的对全身能量消耗的相互作用的作用。

鼠标是目前肥胖症研究的主要模式。短世代间隔，并在鼠标可用的遗传工具已经无可匹敌的日期。然而，斑马鱼也有一个短的世代间隔（3 - 4个月）和甚至超过了鼠标的易用性基因操纵1,2。斑马鱼保持近90％的哺乳动物的基因，而远远超过了鼠标的后代和潜在的遗传和药物屏幕3的使用数量。 为了开发用于肥胖症的研究斑马鱼模型的潜力，试验必须制定调查影响体重调节，包括能源消耗的因素。作为代谢物通过β氧化和三羧酸循环氧处理被消耗和NADH 2就产生了。因此，NADH 2是代谢物的通过代谢途径（代谢物氧化）的磁通的直接指标。在poikilotherms H +泄漏通过线粒体内膜，该脱开NADH 2氧化从ATP合成，是4 - 5比恒温动物4倍以下。因此，在斑马鱼NADH 2是非常紧密通过氧化磷酸化连接于ATP的产生。在此，我们描述了测量NADH 2生产幼虫斑马鱼作为能量消耗5代理的检测。 耗氧量的黄金标准测量的能量消耗。然而，为了最好的利用斑马鱼的高通量的潜力，能量消耗的测定必须是适合于高通量。依赖于一个封闭的腔室循环系统氧消耗系统的吞吐量由可用6室的数目的限制。露天氧气消耗量/ CO 2的生产试验也已在斑马鱼5,7应用。这些开放空气96孔板基D系统适于高吞吐量。不幸的是，与环境进行气体交换限制这些测定的灵敏度。我们最近公布了使用氧化还原指示剂的AlamarBlue 5监视NADH 2的检测中的应用。该测定中克服了吞吐量和共同到氧消耗在斑马鱼的分析灵敏度的限制。 斑马鱼正在成为整个身体能量平衡的研究日益重要的模式。在某种程度上，因为斑马鱼是适合于向前遗传筛选和药物屏幕使用。此外，有针对性的遗传操作，包括敲除和敲入，可以快速应用。之前我们已经表明，这种检测可以与浴药的应用和基因敲除或基因敲除结合起来，以确定化合物的基因，改变代谢率5。此外，该试验被设计用于利用固有的斑马鱼的高通量的优点。 </P&gt;

<table><tbody><tr><td>AlamarBlue</td><td>Fisher Scientific</td><td>10-230-103</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Fine Tip DisposableTransfer Pipette</td><td>Fisher Scientific</td><td>13-711-26</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Fisherbrand Graduated Disposable Transfer Pipette</td><td>Fisher Scientific</td><td>13-771-9AM</td><td>Manufactured by Thermo Scientific</td></tr><tr><td>Black sided clear bottom 96-well plates</td><td>Fisher Scientific</td><td>50-320-785</td><td>Manufactured by E&amp;amp;K Scientific Products Inc.</td></tr></tbody></table>

high throughput danio rerio energy expenditure assay

斑马鱼是一种重要的模式生物与必须使斑马鱼的能量平衡和肥胖的研究中广泛应用模式的潜在先天的优势。斑马鱼的小尺寸允许在胚胎测定以在96或384孔板形式进行，基于吗啉代和CRISPR技术促进易于遗传操作，和药物治疗通过浴的应用是可行的。此外，斑马鱼是理想的正向遗传学屏幕允许新基因的发现。定斑马鱼的相对新颖作为模型肥胖，有必要开发工具，充分利用这些好处。在此，我们描述了一个同时测量数千胚胎斑马鱼的能量消耗的方法。我们已经开发出一种整体动物微孔板平台，其中，我们使用96孔板来分离单个鱼和我们评估使用市售的细胞培养物生存力重新累积NADH 2生产代理的AlamarBlue。在变温动物NADH 2产生和能量消耗之间的关系是紧密相连。这种能量消耗测定创建快速筛选药理学或遗传操作直接改变能量消耗或改变响应于施加的药物（例如，胰岛素增敏剂）的潜力。

测定溶液不会在没有胚胎鱼（图1）增加的荧光。然而，该测定是在井内代谢率的微小变化高度敏感。内一个井的单个鱼产生的信号在1小时内（P &lt;0.0001）显著不同于空白孔中。 图2提供了孵育24小时后通过胚胎鱼产生的信号的视觉表示。对于这张照片的目的明确的双面井使用。然而，黑色双面井优选用于测定性能，以防止来自附近孔的荧光信号的浸出。 由于NADH 2诱导减少的AlamarBlue的是不可逆的，信号累积用的时间。这允许小的变化被放大。要说明的是差异累积，从而增加与时间，我们监测由1诱导至5的鱼在1，2中荧光的相对变化，和4小时（图3）。在鱼的各数/孔的荧光的相对变化显著随时间（P &lt;0.001）增加。更重要的是，因为信号与累积时间，在荧光变化的差异而导致的操纵鱼的数量的大小/孔处于比在1小时温育4小时更健壮。因此，通过增加接触时间，我们可以提高检测与给定的治疗相关的差异的能力。 <img alt="图1" src="/files/ftp_upload/53297/53297fig1.jpg" /> 图噪声对信号的1比较。由1斑马鱼在1小时内产生96孔板可检测的荧光变化。韦尔斯不包含鱼（空白）产生小信号。误差棒代表SEM（N = 5 - 8）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53297/53297fig1large.jpg" target="_blank">请点击此处查看大图</a>这个数字。 <img alt="图2" src="/files/ftp_upload/53297/53297fig2.jpg" /> 图测定板在24小时，2的视觉表示。使用该测定所获得的结果的代表性图片。粉红色的井指示的鱼具有高代谢率，紫井温和代谢率，和蓝色的井低代谢率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53297/53297fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53297/53297fig3.jpg" /> 图3.信号差异扩增随着时间。在通过改变鱼的数量/孔随时间增加所产生的荧光信号的差异。由于信号积累随着时间的推移，荧光变化的幅度发散随时间betweensamples吨帽子不同信号生成的速率。误差棒表示SEM（N = 7）。

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

high-throughput-danio-rerio-energy-expenditure-assay

Nanyang Technological University

Research

JoVE Journal

Biology

High Throughput Danio Rerio Energy Expenditure Assay

12.6K 次观看.  Vanderbilt University Medical Center. Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery. 

视频: High Throughput Danio Rerio Energy Expenditure Assay

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

科研

遗传学

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

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

用于果蝇消费量化的高吞吐量微板馈线分析

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. When selecting sites to deposit their eggs, female flies are capable of ranking the relative attractiveness of their options and choosing the &#34;greater of two goods.&#34; However, most egg-laying preference assays are not practical if one wants to take a systematic genetic screening approach to search for the circuit basis underlying this simple decision-making process, as they are population-based and laborious to set up. To increase the throughput of studying of egg-laying preferences of single females, we developed custom chambers that each can simultaneously assay egg-laying preferences of up to thirty individual flies as well as a protocol that ensures each female has a high egg-laying rate (so that their preference is readily discernable and more convincing). Our approach is simple to execute and produces very consistent results. Additionally, these chambers can be equipped with different attachments to allow video recording the egg-laying animals and to deliver light for optogenetics studies. This article provides the blueprints for fabricating these chambers and the procedure for preparing the flies to be assayed in these chambers.

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

This protocol describes a high throughput assay for testing egg-laying preferences of Drosophila melanogaster at single-animal resolution. This assay provides a simple, efficient, and scalable platform to identify genes and circuit components that control a simple decision-making process.

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

神经科学

<meta charset="utf8"></head><body>刷蜥蜴肝线粒体中的线粒体功能评估

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

<meta charset="utf8"></head><body>使用基于读板器的荧光测定法评估线粒体耗氧量

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

使用基于读板器的荧光测定法评估线粒体耗氧量

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

生物化学

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 ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

高通量

摘要

探索更多视频

此视频中的章节

高脂膳食喂养和高通量三测定在果蝇中的含量

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

用于果蝇消费量化的高吞吐量微板馈线分析

高通量检测，以检查个体产蛋首

使用基于读板器的荧光测定法评估线粒体耗氧量

使用基于读板器的荧光测定法评估线粒体耗氧量

使用基于读板器的荧光测定法评估线粒体耗氧量

果蝇幼虫和成年脑的代谢分析

斑马鱼胚胎的代谢谱分析

高通量

摘要

探索更多视频

此视频中的章节

高脂膳食喂养和高通量三测定在果蝇中的含量

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

用于果蝇消费量化的高吞吐量微板馈线分析

高通量检测，以检查个体产蛋首

使用基于读板器的荧光测定法评估线粒体耗氧量

使用基于读板器的荧光测定法评估线粒体耗氧量

使用基于读板器的荧光测定法评估线粒体耗氧量

果蝇幼虫和成年脑的代谢分析

斑马鱼胚胎的代谢谱分析

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