この作品は、国立衛生研究所によってサポートされていましたF32 NS078958（DTB）とR01 AG033620（BG）が付与されます。

1。フライトテスターを組み立てる<ol><li>リングへの安全な飛行シリンダーチェーンクランプを使用して1スタンド。 （お皿の重量を量るためのシリンダーの下に約3cmのままにしておきます。） （注：我々が使用する飛行シリンダー13.5センチ、直径90センチです。） </li><li>飛行シリンダーの下にミネラルオイルの薄い層で、皿の重さに挿入します。 </li><li>リングへの安全な漏斗をリングクランプを使用して2スタンドとクランプ爪。漏斗の底が飛行シリンダーの上部と同じ高さになるように、漏斗の高さを調整します。 （注：漏斗の先端径は、バイアルが通過落下しないよう、ドロップチューブに入れ、バイアルの外径よりも小さくなければなりません。） </li><li>漏斗の上に落下管を挿入し、爪クランプを使って固定します。 （注：私たちは、長さ25cmで落下管を使用してこの高さから落下飛ぶ含有するバイアルに均一な力を持つすべてのハエの一貫したインク吐出が可能な落下管の内径はスライでなければなりません。バイアルが自由にドロップできるようにバイアルの外径よりもTLY大きくなります。） </li><li>適当な大きさにポリアクリルアミドシート（​​S）をカット。 （注：挿入したシートを除去することを助けるために、幅が飛行シリンダの内周面よりも僅かに小さくあるべきである）。 </li><li>シートにもつれトラップの薄い層を適用します。使用前に1時間放置することができます。 （注：挿入/除去のためのシートを把握することがコーティングされていないシート（約3cm）の上部と下部に十分な余地を残している） </li><li>飛行シリンダ内のポリアクリルアミドシートを挿入します。 </li><li>松のサポートブラケットを使用してカメラトラックを組み立てます。 （注：トラックの底部がレンズをブロックすることなく、カメラをサポートできることを確認してください。図1（b）参照。） </li><li>ストッパーを追加し、所定の位置にねじ込みます。 （注：場所をカメラがパノラマモードで、全体のプラスチックシートを表示することができます場所にストッパー。） </li></ol> 2。走行実験<ol> &lt;李&gt;試験しようとするハエのバイアルを収集します。最良の結果を得るには、せいぜい20ハエ/バイアルを使用しています。 </li><li>優しく落下管とリリースバイアル内に挿入して、抜いて、バイアルの底にハエをタップします。 （注：これは、狭い漏斗開口部に当たるまで、バイアルは、落下管に倒れバイアル漏斗に当たると、ハエは飛行シリンダー内に噴射される。） </li><li>空のバイアルを削除するには落下管を持ち上げます。 （注：同じテストグループのハエの複数のバイアルは、単一のポリアクリルアミドシートにアッセイすることができる私たちは、最大200ハエ（20ハエそれぞれの10バイアル）は一枚のシートでテストされ、容易に画像化することができることがわかります。。 </li><li>プラスチックシートを取り外し、平らな白い面に置きます。 （注：ベンチトップが暗色であれば白のポスターボードを使用することができる。） </li><li>プラスチックシートの上にカメラトラックを組み立てます。カメラは、視野内のシートの頂部および底部の両方を有するように、シートの上に十分に高くあるべきである。 </li><li>目に沿ってカメラをスライドさせます電子軌道のパノラマ画像を取得する「捕捉」ボタンを押したまま。 </li><li>油に着陸ハエの数は、各試験のために手動でカウントしてもよい。 </li><li>繰り返しは、指定された実験ではすべての条件のための2.2から2.7を繰り返します。ハエは、各試行間のシートから取り外すことができます。あるいは、いくつかのシートを各試験のために新しいシートを、使用することができる。 </li></ol> 3。データ収集<ol><li> ImageJソフトウェアを使用して開いている画像ファイル。 </li><li>クロップ画像は着陸表面積を含める必要がある場合。 （これはもつれトラップにコーティングされた領域です。） </li><li> 8ビットグレースケールに画像を変換します。 </li><li>白い背景をフィルタリングする「しきい値」を作成します。 （画像→→スレッショルドを調整）。 </li><li> 「粒子を分析」メニューを使用して、各ハエを識別するためにパラメータを設定します。 粒子を識別するために使用するパラメータを定義します（粒子を分析→分析）。私たちは、セットアップにより、我々は見つけることが使用して5〜90ピクセル2および0.4〜1.0の円形の領域を正確にすべてのサンプルを識別します。 </li><li>各粒子の座標の生成されたリストを使用して、各ハエの位置を測定します。 x座標（ピクセル単位）は、着陸の高さを計算するためにセンチメートルに変換することができる。 </li><li> （Microsoft Excelなど）スプレッドシートにテーブルをインポートします。 </li></ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, 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=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., 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=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

著者らは、開示することは、特別の利害関係はありません。

ここに記載される方法を用いて、我々は、以前に可能であったよりも高い効率を提供し、迅速にショウジョウバエの変異体の多数の飛行性能を評価することができた。我々の実験のために、我々は日常的に男性と女性とを分離し、翼を損傷する可能性が攻撃を制限するために、低濃度（20未満のハエ/バイアル）でそれらを発生させます。もう一つの重要な考慮事項は、遺伝的背景の違?...

ショウジョウバエは、長い行動1の遺伝的基礎を研究するために使用されており、研究者は行動2-6の様々なタイプを分析するための多数の方法を考案した。ハエは、神経筋疾患7の有用なモデルを提供するのに特に有用であった。運動行動を研究するために使用される一般的なアッセイは、飛行性能である。オリジナルの飛行試験機は、飛行欠損変異株を識別するための飛行能力1の定量的評価に有用であるが、それはハイスループットスクリーニングへの応用を制限するいくつかの欠点があります。油でコーティングされたシリンダーの使用は面倒で煩雑であるなどの特定の機能を可変力を有するチューブ内にシリンダとハエの導入の長さは、定量的な精度を低下させると、それは、テスタからのライブハエを回収することは困難である。これらの制限を克服するために、我々は多数の改良を含むように飛行試験を変更している。私たちは、 &quot;ドロップTUを追加しました実験とユーザ間のばらつきを排除するためにハエを導入すること」である。我々は、簡単にクリーンアップし、個々のハエの回復を可能にする接着剤を塗布した取り外し可能なアクリルシートを使用して我々は、定量的な精度と信頼性を向上させるために、飛行管の長さを増加して、最終的に、我々は、ハエの着地高さを計算するために、デジタルカメラと画像処理ソフトウェアを使用しています。我々は、これらの改善は、飛行性能に欠陥のための大規模な遺伝子スクリーニングを実施することに興味の任意の研究室に有用であると考えています。

<table border="0" cellpadding="0" cellspacing="0" width="704">
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</table>

an improved method for accurate and rapid measurement of flight performance in drosophila

ショウジョウバエは、飛行を含む行動の分析に有用なモデル系であることが証明されている。最初の飛行試験機は、油でコーティングされたにハエを落とす関与メスシリンダーに、着陸の高さは、はるかにハエがシリンダーの壁と接触するために十分な推力を発生する前に落ちるか評価することにより、飛行性能の尺度を提供した。ここでは、4の主要な改善と飛行試験機の更新版について説明します。まず、我々はすべてのハエがユーザー間でのばらつきをなくし、試験間で類似の速度で飛行シリンダーに入ることを保証するために、「落下管」を追加しました。第二に、我々はもつれトラップ、生きた昆虫を捕獲するよう​​に設計され、接着剤でコーティングされた取り外し可能なプラスチックシートにオイルコーティングを取り替えた。第三に、我々は、飛翔能力のより正確な識別を可能にするために、より長いシリンダーを使用しています。第四に、我々は飛行性能の採点を自動化するために、デジタルカメラと画像処理ソフトウェアを使用しています。これらの改良は、ラップを可能にID、飛行挙動の定量的評価、大規模なデータセットおよび大規模な遺伝子スクリーニングに便利。

<html> 図1Aは、更新された飛行試験器アセンブリの概略図を示す。 図1Bは、トラックの設計がカメラの視野を遮断することなく、パノラマ画像を撮影できるように示している。代表的な結果は、欠陥8-10公知のフライトを有するもどかしい変異体ハエの飛行性能は、野生型カントン-Sハエと比較される図2に示されている。コントロール?...

Watch this Scientific Journal Video about An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila at JoVE.com

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

ここでは、ハイスループットスクリーニングを可能にする、 ショウジョウバエの飛行性能を迅速かつ正確に測定するための方法を記載している。

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 ...

an-improved-method-for-accurate-rapid-measurement-flight-performance

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JoVE Journal

Biology

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

11.6K ビュー.  University of Wisconsin-Madison. ここでは、ハイスループットスクリーニングを可能にする、 ショウジョウバエの飛行性能を迅速かつ正確に測定するための方法を記載している。 

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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

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 ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

ショウジョウバエの視覚と嗅覚を介したフライトコントロールを調査するために磁気テザーシステム

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Visually Mediated Odor Tracking During Flight in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Additionally, recent advances in olfactory molecular genetics and neurophysiology have motivated novel quantitative behavioral analyses to assess the behavioral influence of (e.g.) genetically inactivating specific olfactory activation circuits. We modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. Here we focus on experiments that can be performed after a fly is tethered and is able to navigate in the magnetic arena. We show how to acquire video images optimized for measuring body angle, how to judge stable odor tracking, and we illustrate two experiments to examine the influence of visual cues on odor tracking.

Here we describe how to optimize the acquired video image for an olfactory magnetic-tether (OMT) apparatus. We also describe two sample experimental protocols for studying visuo-olfactory fusion.

ショウジョウバエのフライトの間に視覚的に仲介臭気の追跡

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

ロボロリーパインからのRNA分離の改良法（ P. taeda L.）及びその他の針葉樹種

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders such as diabetes and cardiovascular disease 1-3. An important topic of research into these associated health problems involves the role of endurance exercise as a beneficial intervention.
Exercise training is an inexpensive, non-invasive intervention with several beneficial results, including reduction in excess body fat 4, increased insulin sensitivity in skeletal muscle 5, increased anti-inflammatory and antioxidative responses 6, and improved contractile capacity in cardiomyocytes 7. Low intensity exercise is known to increase mitochondrial activity and biogenesis in humans 8 and mice, with the transcriptional coactivator PGC1-&alpha; as an important intermediate 9,10. 
Despite the importance of exercise as a tool for combating several important age-related diseases, extensive longitudinal genetic studies have been impeded by the lack of an endurance training protocol for a short-lived genetic model species. The variety of genetic tools available for use with Drosophila, together with its short lifespan and inexpensive maintenance, make it an appealing model for further study of these genetic mechanisms. With this in mind we have developed a novel apparatus, known as the Power Tower, for large scale exercise-training in Drosophila melanogaster 11. The Power Tower utilizes the flies' instinctive negative geotaxis behavior to repetitively induce rapid climbing. Each time the machine lifts, then drops, the platform of flies, the flies are induced to climb. Flies continue to respond as long as the machine is in operation or until they become too fatigued to respond. Thus, the researcher can use this machine to provide simultaneous training to large numbers of age-matched and genetically identical flies. Additionally, we describe associated assays useful to track longitudinal progress of fly cohorts during training.

Endurance Training Protocol and Longitudinal Performance Assays for Drosophila melanogaster

We describe the first endurance training protocol for an important genetic model species, Drosophila melanogaster, and outline several assays to chart improvements in mobility following training.

キイロショウジョウバエのための持久力トレーニングのプロトコルと縦パフォーマンスアッセイ

One of the most pressing problems facing modern medical researchers is the surging levels of obesity, with the consequent increase in associated disorders ...

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 ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

エキソソームの定量化とサイズの測定のための革新的方法

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代謝率の測定に<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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...