这项工作是由美国国立卫生研究院（K08 - DK073545和赞助NICHD的智力和发育残疾研究中心新研究者奖），费城基金会，宾夕法尼亚大学麦凯布奖（MJF），以及特里斯坦部分经费马伦基金（MJF和MY）。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

<ol>
	<li>Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> C. elegans: A practical approach. , (1999).</li><li>Dingley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+dysfunction+variably+increases+oxidant+stress+in+Caenorhabditis+elegans.">Mitochondrial respiratory chain dysfunction variably increases oxidant stress in Caenorhabditis elegans.</a> Mitochondrion. 10, 125-136 (2010).</li><li>Jones, B. N., Gilligan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=o-Phthaldialdehyde+precolumn+derivatization+and+reversed-phase+high-performance+liquid+chromatography+of+polypeptide+hydrolysates+and+physiological+fluids.">o-Phthaldialdehyde precolumn derivatization and reversed-phase high-performance liquid chromatography of polypeptide hydrolysates and physiological fluids.</a> Journal of chromatography. 266, 471-482 (1983).</li><li>Falk, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+pathway+profiling+of+mitochondrial+respiratory+chain+mutants+in+C.+elegans.">Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans.</a> Molecular genetics and metabolism. 93, 388-397 (2008).</li><li>Yudkoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+in+vivo+ureagenesis+with+stable+isotopes.">Measuring in vivo ureagenesis with stable isotopes.</a> Molecular genetics and metabolism. 100, 37-41 (2010).</li><li>Tsang, W. Y., Sayles, L. C., Grad, L. I., Pilgrim, D. B., Lemire, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+deficiency+in+Caenorhabditis+elegans+results+in+developmental+arrest+and+increased+life+span.">Mitochondrial respiratory chain deficiency in Caenorhabditis elegans results in developmental arrest and increased life span.</a> J Biol Chem. 276, 32240-32246 (2001).</li></ol>

应用质谱测量同位素丰度的中间代谢产物，提供了一次关键的生化改变5奇妙的详细图片。在这里，我们提供详细的协议，利用这个高度敏感和特异的方法，以评估中介代谢通量基因通用的模型动物，C. 线虫 。事实上，饲养动物活体与稳定同位素标记的代谢前体（ 如 13 C -葡萄糖）允许将中介代谢途径流量新颖的见解，测量时在下游代谢产物的同位素富集。我们已经开发出可靠的方法，获得通过糖酵解，丙酮酸代谢，三羧酸循环强大的分析通量。要做到这一点无论是在美联储稳定同位素幼体早期发育阶段的动物，或在有丝分裂后的成年期开始。可以结合全虫游离氨基酸分析研究代谢产物在不同物种的同位素富集高效液相色谱法，如前面所述4，确定在不同物种免费蠕虫丙氨酸，天门冬氨酸，谷氨酸，谷氨酰胺绝对同位素富集。事实上，一贯的同位素富集，是获得在1,000至2,000个年轻的成年同步蠕虫等分测量时，氨基酸和有机酸分析物。此种方法能灵敏地歧视中介通量异常核基因为基础的线粒体突变株与野生型蠕虫。 这项技术拥有价值调查相关的具体生化途径的通量变化常见的代谢与生俱来的错误。与稳定同位素标记的葡萄糖的利用率，特别是通过线粒体疾病相关的中央的代谢途径通知碳通量。事实上，我们的数据表明，应用此种方法能灵敏地歧视中介通量异常核基因为基础的线粒体复合三亚基突变株（ISP - 1（qm150））相对于野生型蠕虫。 重要的是要认识到，由于同位素曝光超过数天完成了一个周期，量化同位素富集代表超过规定的时间内可能是在细胞模型确定的"稳定状态"浓缩的价值，而不是量化通量暴露的同位素几分钟到几小时内。另一个潜在的局限性，对线虫的发展过程中询问途径通量与幼体发育的不同长度，在发生特定的突变株。例如，许多严重的线粒体基因突变是已知的导致第三（L3）的幼虫期 6逮捕。因此，只在动物体内存活到成年的同位素掺入的分析可能无法代表整个突变人口。然而，这种方法可以适用于评估中介代谢产物在一个特定的幼虫阶段（如二级），而不是等待的动物，以达到成年阶段的同位素掺入。同样，进入另一种幼虫阶段可能作为的dauer已知的动物有明显改变（可能降低）代谢通量相对动物进行直接通过四个幼虫。未来的研究也可以进行直接评估在不同遗传背景的dauer阶段动物的同位素掺入。动物暴露到一个固定时间段开始，一旦动物达到年轻的成年阶段（在这里，24至48小时）的稳定同位素（详见协议乙）删除变量发育时期的影响。虽然只询问13的C -葡萄糖糖酵解，丙酮酸代谢和三羧酸循环的通量，其他同位素示踪剂可能被评定为通过其他途径线虫利益的生化途径通量询问。例如，15的N -甘氨酸可能被用来评估线虫氨基酸周转。 这种方法的另一个潜在的局限性代谢物检测的灵敏度水平。我们的经验表明，至少500名年轻的成年线虫需要，这里的HPLC和GC / MS方法，成功地量化同位素掺入。使用仪器具有更大的敏感性，如超高效液相色谱（UPLC），，可能会允许研究的动物数量进一步减少，尽管我们预期这是不可能的，允许可靠的定量的代谢物和同位素掺入的代谢产物种类从单一的蠕虫的。我们研究了蠕虫人口1,000元实验动物，我们发现，可靠地检测每一株低丰度代谢物。然而，如GABA的一些代谢物，只能是可靠的量化线虫更大的人口，秩序1 × 10 6动物4。 总之，稳定同位素分析提供了一种非侵入性和安全（非放射性）的方法，早已被用来研究先天性代谢缺陷。这种方法应用到 C 线虫提供了一种新的在体内，实时，发生在整体动物水平的代谢改变，这可能会导致从个人的遗传性疾病和/或药物剂风险能力进行调查。

<table><tbody><tr><td>S. basal</td><td></td><td>&lt;sup&gt;1&lt;/sup&gt; - page 59</td><td>Protocols A and B</td></tr><tr><td>Cholesterol</td><td>Sigma-Aldrich</td><td>C-8503</td><td>Protocol B</td></tr><tr><td>OP50 &lt;em&gt;E. coli&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td></td><td>Protocols A and C</td></tr><tr><td>K12 &lt;em&gt;E. coli&lt;/em&gt;</td><td>Caenorhabditis Genetics Center</td><td></td><td>Protocol B</td></tr><tr><td>NGM agar</td><td>Research Products International Corp.</td><td>N81800-1000.0</td><td>Protocols A, B, and C</td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C glucose (universally labeled)</td><td>Cambridge Isotope Laboratories</td><td>CLM-1396</td><td>Protocol A and C</td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C glucose (1,6 labeled)</td><td>Cambridge Isotope Laboratories</td><td>CLM-2717</td><td>Protocol B</td></tr><tr><td>60% Perchloric acid (PCA)</td><td>Fisher Scientific</td><td>MK2766500</td><td>Protocols A and B</td></tr><tr><td>&amp;#949;-aminocaproic acid (Internal Standard)</td><td>Sigma-Aldrich</td><td>A-2504</td><td>Protocols A and B</td></tr><tr><td>MTBSTFA</td><td>Regis</td><td>270243</td><td>Protocol E</td></tr><tr><td>Acetonitrile</td><td>Regis</td><td>270010</td><td>Protocol E</td></tr><tr><td>AG 1-X8, 100-200, Chloride form resin</td><td>Bio-Rad</td><td>140-1441</td><td>Protocols A and B (organic acid extraction)</td></tr><tr><td>AG 50W-X8, 100-200, Hydrogen form resin</td><td>Bio-Rad</td><td>142-1441</td><td>Protocols A and B (amino acid extraction)</td></tr><tr><td>NaHCO3</td><td>Sigma-Aldrich</td><td>S-8875</td><td>Protocol C</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S8045</td><td>Protocol C</td></tr><tr><td>3N HCl</td><td>Sigma-Aldrich</td><td>320331</td><td>Protocol D</td></tr><tr><td>1N HCl</td><td>Sigma-Aldrich</td><td>320331</td><td>Protocol A</td></tr><tr><td>0.1 N HCl</td><td>Sigma-Aldrich</td><td>320331</td><td>Protocol D</td></tr><tr><td>4N NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>Sigma-Aldrich</td><td>30501</td><td>Protocol D</td></tr><tr><td>4N KOH</td><td>Sigma-Aldrich</td><td>484016</td><td>Protocols A and B</td></tr><tr><td>Deionized water</td><td>Milli Q Biocel</td><td>ZMQA60F01</td><td>Protocol D</td></tr><tr><td>Helium</td><td>Airgas</td><td>24001364</td><td>Protocol C2</td></tr><tr><td>SMZ-800 Zoom Stereo-Microscope</td><td>Nikon Instruments</td><td>NI MNA41000</td><td>Protocols A, B, and C</td></tr><tr><td>pH meter</td><td>Fisher Scientific</td><td>S68167</td><td>Protocols A and B</td></tr><tr><td>Table top centrifuge - 5804R</td><td>Eppendorf</td><td>05-400-93</td><td>Protocols A and B</td></tr><tr><td>Incubated platform shaker</td><td>New Brunswick Scientific</td><td>M1324-0004</td><td>Protocol B</td></tr><tr><td>Mini LabRoller Rotator</td><td>Labnet International</td><td>H-5500</td><td>Protocols A and B</td></tr><tr><td>Pestles</td><td>Kontes Corp</td><td>K749520-0090</td><td>Protocols A and B</td></tr><tr><td>Drill</td><td>Kontes Corp</td><td>K749540-0000</td><td>Protocols A and B</td></tr><tr><td>1 L glass beaker</td><td>Fisher Scientific</td><td>02-539P</td><td></td></tr><tr><td>1 L Erlenmeyer Flasks</td><td>VWR international</td><td>89000-368</td><td></td></tr><tr><td>25 mL Erlenmeyer Flasks</td><td>VWR international</td><td>89000-356</td><td>Protocol B</td></tr><tr><td>7ml round-bottom glass tubes + rubber stopper</td><td>VWR international</td><td>VT6431</td><td>Protocols A, B, and C1,C2</td></tr><tr><td>10 ml round-bottom glass tubes + rubber stopper</td><td>VWR international</td><td>VT6430</td><td>Protocol C2</td></tr><tr><td>Blue top tubes</td><td>Labco</td><td>438B</td><td></td></tr><tr><td>50 ml conical plastic tubes</td><td>Falcon BD</td><td>14-959-49A</td><td>All protocols</td></tr><tr><td>1.5 ml microfuge tubes</td><td>Fisher Scientific</td><td>02-681-320</td><td>Protocols A and B</td></tr><tr><td>Syringes (1 mL, 10 mL, 20 mL)</td><td>BD Biosciences</td><td>301025, 301029, 301031</td><td>Protocols C1 and C2</td></tr><tr><td>25 gauge needles</td><td>Fisher Scientific</td><td>22-253-131</td><td>Protocols C1 and C2</td></tr><tr><td>Poly-Prep Chromatography Columns</td><td>Bio-Rad</td><td>731-1550</td><td>Protocol D</td></tr><tr><td>Pasteur pipettes</td><td>VWR international</td><td>14673-043</td><td>Protocol D</td></tr><tr><td>60mm Petri dishes</td><td>VWR international</td><td>25373-085</td><td>Protocol C</td></tr><tr><td>Glass chamber (cut bottom of 1 L flask) fitted with grease-sealed 3-way stopcock</td><td>Custom Made</td><td></td><td>Protocol C</td></tr><tr><td>Optically transparent glass plate</td><td>Custom Made</td><td></td><td>Protocol C</td></tr><tr><td>Reacti-Vap III Evaporator</td><td>Thermo Fisher Scientific, Inc.</td><td>18826</td><td>Protocol D</td></tr><tr><td>Cotton</td><td>VWR international</td><td>14224-516</td><td>Protocol D</td></tr><tr><td>High Vacuum Grease</td><td>Dow Corning</td><td>1597418</td><td>Protocol C1</td></tr><tr><td>Aluminum Foil</td><td>Fisher Scientific</td><td>01-213-18</td><td>Protocol B</td></tr><tr><td>Timer</td><td>ISC Bioexpress</td><td>T-2504-5</td><td>Protocol C</td></tr><tr><td>Gas-Ratio Mass Spectrometer</td><td>Thermo Fisher Scientific, Inc.</td><td></td><td>Protocol D</td></tr><tr><td>GC-MS</td><td>Hewlett-Packard</td><td>5980/5971</td><td>Protocol D</td></tr><tr><td>GC-MS</td><td>Agilent Technologies</td><td>6980N/5973N</td><td>Protocol D</td></tr><tr><td>HPLC</td><td>Varian Inc., Agilent</td><td>9010</td><td>Protocol D</td></tr></tbody></table>

stable isotopic profiling of intermediary metabolic flux in developing and adult stage caenorhabditis elegans

稳定同位素分析早已允许在细胞和哺乳动物模型中的基因突变和/或药物疗法的代谢后果的敏感调查。在这里，我们描述了详细的方法来执行的线虫 ，线虫的中间代谢及代谢通量的稳定同位素分析。全虫，氨基酸，标记的二氧化碳，有机酸标记，标记的氨基酸分析中的稳定同位素线虫生长培养基琼脂板或青壮年开始从早期的发展，而暴露在各种药物治疗的动物的描述方法在液体培养。游离氨基酸是由高性能液相色谱法（HPLC）在整个蠕虫分装在4％高氯酸提取量化。通用标记13的C -葡萄糖或1,6 - 13彗星2 -葡萄糖利用稳定同位素前体，其标示碳质谱（大气和溶解二氧化碳）以及在代谢物通量的指示通过糖酵解追溯到丙酮酸代谢，三羧酸循环。代表结果证明野生型线虫同位素照射时间的影响，各种细菌的结算协议，并替代的蠕虫病毒破坏方法，以及同位素掺入在线粒体复合物III突变蠕虫的相对程度（ISP - 1 （qm150） ）相对于野生型蠕虫。生活线虫的稳定同位素分析中的应用提供了一种新颖的能力，以调查在整体动物水平的实时代谢是由个人的遗传性疾病和/或药物治疗引起的改建。

Watch this Scientific Journal Video about Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans at JoVE.com

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

稳定同位素分析气相色谱质谱中介代谢通量的质谱分析中所述的线虫，<em&gt;线虫</em&gt;。详细的评估中的二氧化碳，有机酸，氨基酸和同位素暴露在琼脂平板上的发展，或在液体培养成年的同位素富集的方法。

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

stable-isotopic-profiling-intermediary-metabolic-flux-developing

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

13.4K 次观看.  The Children's Hospital of Philadelphia. 稳定同位素分析气相色谱质谱中介代谢通量的质谱分析中所述的线虫，线虫。详细的评估中的二氧化碳，有机酸，氨基酸和同位素暴露在琼脂平板上的发展，或在液体培养成年的同位素富集的方法。

视频: Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

科研

生物化学

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of diverse human diseases. Worms are particularly well-suited for high-throughput genetic and drug screens to gain deeper insight into therapeutic targets and therapies by exploiting their fast development cycle, large brood size, short lifespan, microscopic transparency, low maintenance costs, robust suite of genomic tools, mutant repositories, and experimental methodologies to interrogate both in vivo and ex vivo physiology. Worm locomotor activity represents a particularly relevant phenotype that is frequently impaired in mitochondrial disease, which is highly heterogeneous in causes and manifestations but collectively shares an impaired capacity to produce cellular energy. While a suite of different methodologies may be used to interrogate worm behavior, these vary greatly in experimental costs, complexity, and utility for genomic or drug high-throughput screens. Here, the relative throughput, advantages, and limitations of 16 different activity analysis methodologies were compared that quantify nematode locomotion, thrashing, pharyngeal pumping, and/or chemotaxis in single worms or worm populations of C. elegans at different stages, ages, and experimental durations. Detailed protocols were demonstrated for two semi-automated methods to quantify nematode locomotor activity that represent novel applications of available software tools, namely, ZebraLab (a medium-throughput approach) and WormScan (a high-throughput approach). Data from applying these methods demonstrated similar degrees of reduced animal activity occurred at the L4 larval stage, and progressed in day 1 adults, in mitochondrial complex I disease (gas-1(fc21)) mutant worms relative to wild-type (N2 Bristol) C. elegans. This data validates the utility for these novel applications of using the ZebraLab or WormScan software tools to quantify worm locomotor activity efficiently and objectively, with variable capacity to support high-throughput drug screening on worm behavior in preclinical animal models of mitochondrial disease.

Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease

This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function.

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

Caenorhabditis elegans is widely recognized for its central utility as a translational animal model to efficiently interrogate mechanisms and therapies of ...

行为学

测量 秀丽隐杆 线虫食物摄入量和寿命的高通量测定

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

作者聚焦：探索脂毒性与 HFpEF 之间的关系 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

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

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 obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

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

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

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 ...

生物学

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

摘要

探索更多视频

此视频中的章节

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

生化和高通量的微观评估的脂肪含量

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

可筛选的

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

摘要

探索更多视频

此视频中的章节

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

线虫线虫中乳酸和丙酮酸细胞的小尺度比色测定

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

线粒体疾病脑 膜炎基因 模型中量化动物活动的实验方法比较分析

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

生化和高通量的微观评估的脂肪含量

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

可筛选的

脂肪酸¹³C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

线粒体疾病脑膜炎基因模型中量化动物活动的实验方法比较分析

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量