Name: measurement of metabolic rate in drosophila using respirometry
Uploaded: 2014-06-24T15:00:00
Description: Watch this Scientific Journal Video about Measurement of Metabolic Rate in Drosophila using Respirometry at JoVE.com

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?...

<ol>
	<li>Van Voorhies, W. A., Khazaeli, A. A., Curtsinger, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+the+&amp;#34;rate+of+living&amp;#34;+model:+further+evidence+that+longevity+and+metabolic+rate+are+not+inversely+correlated+in+Drosophila+melanogaster.">Testing the &amp;#34;rate of living&amp;#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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-specific+decrease+in+aerobic+efficiency+associated+with+increase+in+oxygen+free+radical+production+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+contribution:+long-lived+Drosophila+melanogaster.+lines+exhibit+normal+metabolic+rates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+modulates+metabolic+rate+and+temperature+sensitivity+in+Drosophila+melanogaster.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+temperature+preferences+and+energy+homeostasis+in+dystroglycan+mutants.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+muscular+dystrophy:+a+genetic+screen+for+dystroglycan+and+dystrophin+interactors+in+Drosophila.+identifies+cellular+stress+response+components.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperthermic+seizures+and+aberrant+cellular+homeostasis+in+Drosophila+dystrophic.+muscles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+and+metabolism+in+Drosophila+melanogaster:+genetic+correlations+between+life+span+and+age-specific+metabolic+rate+in+populations+artificially+selected+for+long+life.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+equivalents+of+CO2+and+their+importance+in+assessing+energy+expenditure+when+using+tracer+techniques.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epicurean+fly:+using+Drosophila+melanogaster.+to+study+metabolism.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+flies+and+men:+insights+on+organismal+metabolism+from+fruit+flies.">Of flies and men: insights on organismal metabolism from fruit flies.</a> BMC Biology. 11, 38 (2013).</li></ol>

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。

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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

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 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 ...

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster

The power of Drosophila genetics is increasingly being applied to questions of hormone signaling and metabolism and to the development of models of human disease in this organism. Sensitive methods for measurements of parameters such as metabolic rates are needed to drive the understanding of physiology and disease in small animals such as the fruit fly. The method described here assesses fuel oxidation in small numbers of adult flies fed food containing trace amounts of 14C-labeled substrates such as glucose or fatty acid. After the feeding period and any additional experimental manipulations, flies are transferred to short tubes capped with mesh, which are then placed in glass vials containing KOH-saturated filter paper that traps exhaled, radiolabeled CO2 generated from oxidation of radiolabeled substrates as potassium bicarbonate, KHCO3. This radiolabeled bicarbonate is measured by scintillation counting. This is a quantitative, reproducible, and simple approach for the study of fuel oxidation. The use of radiolabeled glucose, fatty acids, or amino acids allows determination of the contribution of these different fuel sources to energy metabolism under different conditions such as feeding and fasting and in different genetic backgrounds. This complements other approaches used to measure in vivo energy metabolism and should further the understanding of metabolic regulation.

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.

放射性標識基質からの二酸化炭素生成での測定<em&gt;キイロショウジョウバエ</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 ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

腸ホルモン分泌の研究と腸内分泌細胞の生細胞イメージングを対象ネズミ小腸の混合初代培養

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.