我々は我々の研究の資金調達のためのマックス·プランク協会に感謝したいと思います。

呼吸計の1。準備<ol><li> 50μlのキャピラリーマイクロピペットの挿入を可能にするためにカミソリの刃で千μlのピペットチップをカットし、できるだけまっすぐピペットチップを取得しよう。 </li><li>ピペットに泡の部分を置き、ピペットチップでそれを押し下げます。 </li><li> CO 2吸収剤を少量添加すると、発泡体の二枚で、それが含まれています。 </li><li>マイクロピペットをピペットチップに挿入されている場所で、接着剤を適用します。 </li><li>接着剤を乾燥させるために、一晩呼吸のままにしておきます。 呼吸計の概略図を図1Aに示されている。 </li></ol>測定所の2。準備<ol><li>目に見えるカラー化になります比でエオシンで水を混合することにより、チャンバ溶液を調製する。 </li><li>チャンバー内にエオシン/水溶液を注ぐ。 </li><li>センチメートルスケールで、チャンバの側面の1にラベルを付けます。 </li></ol><ol><li>マーカーで、個々の呼吸計にラベルを付けます。 </li><li> 2を COと各呼吸計内部の希望する遺伝子型の3-5ハエを配置する別の方法を使用してハエを麻酔。 </li><li>粘土のパテを使用して、上部にしっかりと呼吸計を密封する。 </li><li>ハエは約15分間麻酔から回復することができます。 </li><li>大気中のコントロールとして使用される、ハエなしで1呼吸を準備します。 </li></ol> 4。実験を行う<ol><li>チャンバーの上部に上下に開いて1.5ミリリットルエッペンドルフチュー​​ブホルダーを取り付けることにより、チャンバ内の呼吸計を掛ける。 </li><li>ダウン先端が着色した溶液に浸すことを可能チャンバ内へのマイクロピペットチップで呼吸計を挿入します。 </li><li>温度および圧力fluctより強い分離を提供する蓋カバーとチャンバーとの間にワセリンを追加uations。 </li><li>ふたを閉め、システムが15分間平衡化することができます。 </li><li> （ 図1Bに示す例を参照）、各マイクロピペット内の液体のレベルが表示されているので、スケールがあることを確認して、チャンバの写真を撮る。 </li><li> 1〜2時間後、別の写真を撮る。 </li><li>実験が終了すると、呼吸計からハエを削除し、必要に応じて、重さや、さらに必要に応じてバイアルに戻ってそれらを転送します。 </li></ol>結果5。分析<ol><li>オープンImageJソフトウェア11を使用して画像を取得しました。 </li><li>各写真にスケールを使用して、ソフトウェアでピクセルスケーリングを設定します。 </li><li>液体は初め（D1）で撮影された画像内の決定された基準点から移動した距離（ΔD）と実験終了（D2）を測定します。概略的な例を図1Cに示されている。 </li><li>式で（μL/ HR /飛ぶ）生成されるCO 2の量を計算します。 </li></ol><img alt="図1" fo:content-width="2in" src="/files/ftp_upload/51681/51681eq1.jpg" />センチメートルでマイクロピペット管のRは=半径 液体はセンチメートル単位で測定された試験試料のマイクロピペットで上に移動したΔD=距離 液体が（ハエなし）陰性対照試料のマイクロピペット中に移動した距離=Δcを 使用ハエのN =数 H =時間

<ol><li>Van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Testing+the+%26%2334%3Brate+of+living%26%2334%3B+model%3A+further+evidence+that+longevity+and+metabolic+rate+are+not+inversely+correlated+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Testing the &#34;rate of living&#34; model: further evidence that longevity and metabolic rate are not inversely correlated in Drosophila melanogaster.</a> J Appl Physiol. 97, 1915-1922 (2004).</li>
<li>Ross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-specific+decrease+in+aerobic+efficiency+associated+with+increase+in+oxygen+free+radical+production+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Journal+of+Insect+Physiology%22%5BJournal%5D)">Age-specific decrease in aerobic efficiency associated with increase in oxygen free radical production in Drosophila melanogaster.</a> Journal of Insect Physiology. 46, 1477-1480 (2000).</li>
<li>van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selected+contribution%3A+long-lived+Drosophila+melanogaster.+lines+exhibit+normal+metabolic+rates%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Selected contribution: long-lived Drosophila melanogaster. lines exhibit normal metabolic rates.</a> J Appl Physiol. 95, 2605-2613 (2003).</li>
<li>Hulbert, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+rate+is+not+reduced+by+dietary-restriction+or+by+lowered+insulin%2FIGF-1+signalling+and+is+not+correlated+with+individual+lifespan+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Experimental+Gerontology%22%5BJournal%5D)">Metabolic rate is not reduced by dietary-restriction or by lowered insulin/IGF-1 signalling and is not correlated with individual lifespan in Drosophila melanogaster.</a> Experimental Gerontology. 39, 1137-1143 (2004).</li>
<li>Ueno, T., Tomita, J., Kume, S., Kume, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+modulates+metabolic+rate+and+temperature+sensitivity+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Dopamine modulates metabolic rate and temperature sensitivity in Drosophila melanogaster.</a> PLoS ONE. 7, (2012).</li>
<li>Takeuchi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+temperature+preferences+and+energy+homeostasis+in+dystroglycan+mutants%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Changes in temperature preferences and energy homeostasis in dystroglycan mutants.</a> Science. 323, 1740-1743 (2009).</li>
<li>Kucherenko, M. M., Marrone, A. K., Rishko, V. M., Magliarelli Hde, F., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stress+and+muscular+dystrophy%3A+a+genetic+screen+for+dystroglycan+and+dystrophin+interactors+in+Drosophila.+identifies+cellular+stress+response+components%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Stress and muscular dystrophy: a genetic screen for dystroglycan and dystrophin interactors in Drosophila. identifies cellular stress response components.</a> Developmental Biology. 352, 228-242 (2011).</li>
<li>Marrone, A. K., Kucherenko, M. M., Wiek, R., Gopfert, M. C., Shcherbata, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperthermic+seizures+and+aberrant+cellular+homeostasis+in+Drosophila+dystrophic.+muscles%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Hyperthermic seizures and aberrant cellular homeostasis in Drosophila dystrophic. muscles.</a> Scientific Reports. 1, 47 (2011).</li>
<li>Khazaeli, A. A., Van Voorhies, W., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Longevity+and+metabolism+in+Drosophila+melanogaster%3A+genetic+correlations+between+life+span+and+age-specific+metabolic+rate+in+populations+artificially+selected+for+long+life%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Longevity and metabolism in Drosophila melanogaster: genetic correlations between life span and age-specific metabolic rate in populations artificially selected for long life.</a> Genetics. 169, 231-242 (2005).</li>
<li>Elia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+equivalents+of+CO2+and+their+importance+in+assessing+energy+expenditure+when+using+tracer+techniques%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Energy equivalents of CO2 and their importance in assessing energy expenditure when using tracer techniques.</a> The American Journal of Physiology. 260, (1991).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+an+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li>
<li>Bharucha, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+epicurean+fly%3A+using+Drosophila+melanogaster.+to+study+metabolism%5BTitle%5D)+AND+%22Pediatric+Research%22%5BJournal%5D)">The epicurean fly: using Drosophila melanogaster. to study metabolism.</a> Pediatric Research. 65, 132-137 (2009).</li>
<li>Rajan, A., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+flies+and+men%3A+insights+on+organismal+metabolism+from+fruit+flies%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">Of flies and men: insights on organismal metabolism from fruit flies.</a> BMC Biology. 11, 38 (2013).</li>
</ol>

我々は、開示することは何もありません。

このプロトコルでは、ハエにおけるCO 2産生を測定するための安価で信頼性の高い方法を記載している。我々は、この実験が容易であることが見出さ行うことが迅速かつ他の研究1,6,9と一致して再現性のあるデータを生成する。ここで概説したプロトコルは、簡単に任意の研究室の予算や利用可能な材料に合わせて変更することができます。各個々の呼吸の構成であれば、チャ...

生化学クレブス回路は、CO 2を生成する炭水化物、脂肪、およびタンパク質由来の酢酸酸化によってATPを生成します。 ショウジョウバエでは、O 2入力は直接CO 2の出力と相関し、代謝1のレベルを反映している。これにより、CO 2の出力の測定は、正常老化と代謝2-5に関連研究において使用されている。ここに私たちの研究室では、任意の特別な装置を必要とせずに、最大18サンプル中のCO 2産生を測定することができるように、あらかじめ設計された実験装置を変更しました。その他、我々は以前に筋ジストロフィー関連タンパク質、ジストログリカン（DG）6-8が不足しているハエの代謝率の差を表示するには、このメソッドを使用している。 O酸化的代謝に用いられる2は、呼吸廃棄物として排出されるCO 2に変換される。建設手作りの呼吸計が消費されたTiON O 2の割合の決定を可能にすることが記載されている。ハエを効率的に気相からそれを排除し、CO 2排出された吸収する物質を密閉容器内に配置される。ガス容積（減圧）の変化は、閉鎖呼吸計に取り付けられたガラス毛細管内の流体の変位によって測定される。 他のものよりもこの技術の主な利点はコストです。これまでの研究では、ガス分析と技術的に高度な呼吸計測システム1,9を使用して、ショウジョウバエによるCO 2産生を測定した。より複雑な機器にもかかわらず、ここで説明する方法の感度が報告された値（ 表1）と同様である。さらに、いくつかの他のグループは、 ショウジョウバエ 4-6の相対的な代謝率を決定するために、この技術のバリエーションを使用している。したがって、このアッセイはreliabを生成するために使用することができるル、任意の実験室で設定でき、教育の目的に使用することができる特殊な機器の購入なしに、ショウジョウバエの代謝に関連する再現性のあるデータ。 一般に、生物の代謝を決定するための技術が受け入れを製造CO 2を測定することで、O 2を消費し、または両方3,4,9。しかし、それは、O 2 1当量のCO 2 1当量を生成すると仮定することができ、生成されたCO 2の正確な比は、10を利用代謝基質に依存する。従って、正確にエネルギ単位での代謝速度を決定するためには、O 2消費およびCO 2が生成さの両方を測定する必要がある。このため、ここで説明する方法は、動物ではなく絶対値とCO 2生成の違いを比較するに特に関連のある。私たちの技術は、ティムの期間にわたって複数の動物のCO 2の生産を統合したがって、E（1〜2時間）とは、動物の活動の平均値を返します。実験動物は、測定対照動物よりも活性であることを信じる理由がある場合には異なるレベルの活性を反映し、必ずしも代謝でした。

<table><tbody><tr><td>BlauBrand IntraMark 50 &amp;micro;l micropipettes</td><td>VWR</td><td>612-1413</td><td></td></tr><tr><td>Soda Lime</td><td>Wako</td><td>CDN6847</td><td></td></tr><tr><td>Eosine&amp;nbsp;</td><td>Sigma</td><td>031M4359</td><td>Any dye that can create visible colorization of liquid can be used</td></tr><tr><td>Thin Layer Chromatorgaphy (TLC) Developing Chamber</td><td>VWR</td><td>21432-761</td><td>Any transparent glass chamber that can be closed with the lid</td></tr><tr><td>Anesthetizer, Lull-A-Fly Kit</td><td>Flinn</td><td>FB1438</td><td></td></tr><tr><td>Power Gel Glue</td><td>Pritt</td><td></td><td></td></tr><tr><td>1 ml pipett tips</td><td>Any</td><td></td><td></td></tr><tr><td>Foam</td><td>Any</td><td></td><td></td></tr><tr><td>Plaesticine Putty</td><td>Any</td><td></td><td></td></tr><tr><td>Scalpel</td><td>Any</td><td></td><td></td></tr><tr><td>Tweezers</td><td>Any</td><td></td><td></td></tr></tbody></table>

measurement of metabolic rate in drosophila using respirometry

代謝障害は、人の健康に影響を与え、頻繁に問題となっている。そのため、代謝を調節するメカニズムを理解することは非常に重要な科学的な課題である。ヒトでの多くの疾患の原因遺伝子は、 ショウジョウバエの異なる障害の発症に関与するシグナル伝達経路を研究するための良いモデル作り、フライホモログを持っている。さらに、 ショウジョウバエの扱いやすには、代謝を調節することができる新たな治療標的を同定するのを助けるために遺伝子スクリーニングを簡素化します。このようなスクリーンを行うために、ハエの代謝状態の変化を同定するための簡単​​かつ迅速な方法が必要である。一般に、二酸化炭素生成は基質酸化及び代謝状態に関する情報を提供するエネルギー消費量の良好な指標である。このプロトコルでは、ハエからのCO 2の出力を測定するための簡単な方法をご紹介します。この技術は、潜在的に代謝速度に影響を与える遺伝的摂動の識別を助けることができる。

メソッドが敏感であることを示すために、我々は、野生型（ オレゴンR）からのCO 2の生産は、男性は18、25、および29℃で飛んで測定され、DGと変異体を飛ぶ。ハエは、25℃で昇温し、測定前に5日間実験温度にシフトした。この外温性種について予想されるように、CO 2の量は、温度（ 図2）に増加した産生した。我々は過去に、砂糖を含まない食事は?...

Watch this Scientific Journal Video about Measurement of Metabolic Rate in Drosophila using Respirometry at JoVE.com

Measurement of Metabolic Rate in Drosophila using Respirometry

代謝障害はヒトにおいて最も一般的な疾患の1つである。遺伝的に扱いやすいモデル生物D.キイロは、代謝を調節する新規遺伝子を同定するために使用することができる。本稿では、そのCO 2産生を測定することにより、ハエに代謝率を研究することができ、比較的簡単な方法を説明します。

代謝率の測定に<em&gt;ショウジョウバエ</em&gt;呼吸計測を使用した

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

measurement-of-metabolic-rate-in-drosophila-using-respirometry

Research

JoVE Journal

Biology

Measurement of Metabolic Rate in Drosophila using Respirometry

19.6K ビュー.  Max Planck Institute for Biophysical Chemistry. 代謝障害はヒトにおいて最も一般的な疾患の1つである。遺伝的に扱いやすいモデル生物D.キイロは、代謝を調節する新規遺伝子を同定するために使用することができる。本稿では、そのCO 2産生を測定することにより、ハエに代謝率を研究することができ、比較的簡単な方法を説明します。 

ビデオ: Measurement of Metabolic Rate in Drosophila using Respirometry

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral paradigm, the experimenter usually assumes that the rapid diffusion of odorant molecules from the source leads to the creation of a stable gradient in the dish. To establish a quantitative correlation between sensory inputs and behavioral responses, it is necessary to achieve a more thorough characterization of the odorant stimulus conditions. In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

In this video article, we describe a new method allowing the construction of odorant gradients with stable and controllable geometries. We briefly illustrate how these gradients can be used to screen for olfactory defects (full and partial anosmia) and to study more subtle features of chemotaxis behavior.

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

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.

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

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

生化学

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.

キイロショウジョウバエ幼虫と大人の脳の代謝解析

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

神経科学

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

における寿命の測定<em&gt;キイロショウジョウバエ</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

飛行性能の正確かつ迅速な測定のための改善された方法での<em&gt;ショウジョウバエ</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活性の測定<em&gt;ショウジョウバエ</em&gt;アクティビティモニタ

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

ショウジョウバエの微小呼吸器測定法の適応 - CASK変異体代謝への洞察

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.

著者スポットライト:ショウジョウバエのメラノガスターと砂漠に適応したカブトムシに対する温度の影響 

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.

電量マイクロレスピロメトリーを用いたショウジョウバエのメラノガスターにおけるO2消費量の測定

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

代謝率の測定に

要約

さらに動画を探す

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

ショウジョウバエのティッシュの最小限の量を使用してのグリセリンの繊維でミトコンドリアの酸素消費量の測定

キイロショウジョウバエ幼虫と大人の脳の代謝解析

における寿命の測定

意識マウスにおける糸球体濾過率の測定のためのハイスループット法

飛行性能の正確かつ迅速な測定のための改善された方法での

幼虫活性の測定

外酸生産の測定と解析は、解糖速度を決定するために

電量マイクロレスピロメトリーを用いたショウジョウバエのメラノガスターにおける_O2消費量の測定

電量マイクロレスピロメトリーを用いたショウジョウバエのメラノガスターにおける_O2消費量の測定