国立科学財団からMFへの助成金によって賄わ

OMTは、仮想プルームシミュレータ2を組み込むために設計された磁気テザーシステム1の適応です。次のプロトコルは、取得されたビデオ画像（その1）を最適化し、基本的なvisuo -嗅覚の実験（第2＆3）の2つのタイプを実行する方法を説明します。 パート1：ビデオの取得ビデオ画像を最適化すると、取得したデータの品質にとって重要です。の少ない画像の次の手順を実行します。 <ol><li>ピンセットを使ってアリーナで新鮮なテザーフライを置きます。 </li><li>カメラビューは、テ​​ザーハエの腹側面に、ガラス管を介して、チャンバーを介して直接上向きになるように透明なアクリルの真空チャンバーの下の位置のFireWireボードカメラ（火- I 1394store.comを）。ビデオシステムのレンズにはIRフィルターコーティングがないことを確認してください。 </li><li>フライを照らす、LEDパネルのすぐ下に置か赤外線LED（小型部品株式会社）の円形配列を使用してください。それぞれが個別に均一な照明のためのフライのボディにLEDが焦点。 </li><li>パネルのシステムからのまぶしさを軽減し、その場で観測された視覚的な手掛かりを最小限に抑えるために、それは平らな黒い塗料でカメラのビュー内のすべての反射面のコートに便利です。室内の照明は、フライアイでは検出まぶしさと明るさ調節を減らすためにオフに切り替える必要があります。 </li><li>視覚的にカメラ画像を検査する。ハエは黒い背景に明るい白色にする必要があります。画像がぼやけている場合、レンズは、焦点が必要になることがあります。ハエが暗い場合、LEDアレイは、焦点の定まらないまたは不十分に適切な照明用電源があります。背景が明るいオブジェクトが含まれている場合、背景の反射率を最小限に抑えるためにだけその場の上に黒のフロックの紙のディスクをマウントします。 </li><li>我々は、Image Acquisition Toolboxを使用して、MATLABで記述されたカスタムサブルーチンでビデオを取得する。 </li></ol>パート2：臭羽毛を見つけるフライ OMTの1つの簡単な実験では、位置空腹（飢餓）フライを観察し、積極的に魅力的な食べ物の匂いのプルームを追跡しています。視覚的な環境を操作すると、確実に食欲の食品の匂いを追跡するハエの能力に影響を与えます。実験は、一般的に、実験の順序で任意のバイアスを最小限に抑えるために、ランダム、ブロック形式で実行されることに注意してください。すべての外部ハードウェアは、（ガスのマルチプレクサ、LEDの舞台、映像の取得）のMatlab（Mathworks社）で記述されたカスタムソフトウェアルーチンによって制御されます。 <ol><li>ピンセットを使ってアリーナで新鮮なテザーフライを置きます。 </li><li>すべての実験の前に、視覚的な表示を使用して、全体360のヨー軸周りの滑らかな動きを確保するためにいくつかの革命のためのフライ3を回転させます。フライが回転しない場合はスムーズにそれを捨てる。問題が解決しない場合、考えられる原因は、位置がずれて磁石です。注：スピンの試験中にいくつかの軌道に向かって競技場の分布が著しいピーク（すなわち、360度以上のフラットではない）表示された場合、位置がずれて磁石に起因する方向のバイアスがある可能性がある。 </li><li>ランダムな順序で、次のサンプルの実験条件でフライを紹介：私水蒸気、臭気のポートから放射されると）、高コントラストの縞模様、ⅱ）魅力的な臭気蒸気で高コントラストな縞模様、ⅲ）単一の垂直のストライプが90度をオフセット水蒸気のノズルから、およびiv）臭気の蒸気を持つ単一の90度オフセットストライプ。我々の経験では、30秒間隔では、ハエが独自に臭気源を見つけるために挑戦されている実験のためにうまく働く。我々は（ほとんどの有用な組み込みMATLAB関数のいずれかを各実験条件の期間中、30Hzで映像を取得し、オフラインでのMatlabで書かれたカスタムソフトウェアルーチンを使用して画像を分析する楕円の幾何学的特性を分析regionprops.mです。 </li><li>匂いがオフされた後に高い臭気濃度を使用する場合は、残留臭気は臭気のポートから放出される可能性がある。すべての影響を最小限に抑えるために、これは、システムが任意の残臭をフラッシュできるように回転するパノラマで数秒間その場を回転させると便利ですがあります。 </li><li>それは維持（i）を視覚的に実験的な治療の間にフライを回転させることも有益です積極的に実験に及び（ii）1つの場所に残っているからハエを保つに従事して飛ぶ。 、ⅱ）5秒の中間スピン、III）は20番目の条件のペア＃1、IV）5秒の中間スピン、V）は20番目の条件のペア＃2 I）第二十二初期診断スピン：実験例では、次の順序を持​​つことができます。 </li></ol>パート3：臭羽毛内に残っているハエ OMTにおける第2の基本実験は視覚的に臭羽毛にフライをドラッグすると、瞬時に視覚的な条件を変更してから、プルームのままにその能力を測定する。もう一度、すべての実験はランダムブロックフォーマで行われていることに注意してくださいtは、実験の順序で任意のバイアスを最小限に抑えます。 <ol><li>ピンセットを使ってアリーナで新鮮なテザーフライを置きます。 </li><li>視覚的に動物が（ 第2.2を参照）すべての方向の向きに能力を持っていることを確認するための診断チェックなど、すべての実験の前に（スピン）アリーナの周りフライを数回回転させる。 </li><li>各条件の前に、実験的な臭気（水または酢）をオンにし、視覚的にプルームの位置に小さな縦のストライプを発振することによりプルームにフライを&quot;ドラッグ&quot;。これは、ハエ[4]で観測された強力なストライプの固定の動作を活用します。 </li><li>今ハエがプルームに向かって配向していること、振動のストライプを削除し、次のサンプルビジュアルな条件を提示：水蒸気（ 図1A）を持つ私は）、高コントラストパノラマのストライプ、ⅱ）水蒸気（ 図1B）を持つ一様な背景。 ⅲ）匂い蒸気（ 図1C）、高コントラストのパノラマストライプ、IV）臭気、蒸気（ 図1D）を持つ一様な背景が。 2.3のようにビデオを取得する。第二十二条件のペア＃2 V）、ⅱ）5秒のプル​​ームのストライプ発振; III）は20番目の条件のペア＃1、IV）5秒のプル​​ームのストライプの発振i）は第二十二初期診断スピン：実験例では、流れるような順番があるかもしれません。 </li></ol>代表的な結果： <img src="/files/ftp_upload/old/1110_2009_1_22_1/Fig 1.png" alt="図1" width="600" height="195" /> 図1 第1部では、OMTのビデオ画像を最適化する方法を説明します。ビデオ画像が暗い黒い背景に照らされたその場での明確な見解を持つ必要があります。過度に明るい画像は赤外LEDの輝度を減少させる、バックグラウンドの干渉を低減することにより、または室内の照明をオフにすることで改善することができます。フライが不適切に点灯している場合は、IRのLEDまたはIR LEDの変調強度のフォーカスを調整することにより問題が解決する可能性があります。 パーツ2＆3は、OMTの適切な動作を検証するために実施する簡単な実験を記述する。すべてのケースでは、ハエが視覚的に動きのスムーズかつ完全な範囲を確保するために競技場で数回転を回転させる（微細運​​動応答を経由して）&quot;強制&quot;する必要があります。 第2部ではフライが独自に臭羽毛を見つけるために挑戦されています。実験に従事しているハエを保つために、残留臭気の放出の影響を減らすために、実験を通して見出し、単一の維持からハエを保つために、それは視覚的に試験の間に数秒間その場を回転させると便利です。フライは、かなりの程度に水蒸気の噴煙をローカライズすることはないはず、と常に豊富なパノラマの視覚的手がかりの存在下で臭羽毛をローカライズする必要があります。 パート3ではフライは、視覚的に臭羽毛に直接ドラッグして、その静止視覚的背景の多様性に対するプルーム内の見出しを維持するために要求されます。フライは、水蒸気の煙にドラッグされた場合、それはすぐに離れて回してください。ハエが臭羽毛にドラッグするとロバストなトラッキングを仲介するのに十分な視覚的な手がかりがある場合は、ハエはプルームのままになります。

<ol>
	<li>Bender, J. A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+stimulation+of+saccades+in+magnetically+tethered+Drosophila.">Visual stimulation of saccades in magnetically tethered Drosophila.</a> J Exp Biol. 209, 3170-3182 (2006).</li><li>Duistermars, B. J., Frye, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossmodal+visual+input+for+odor+tracking+during+fly+flight.">Crossmodal visual input for odor tracking during fly flight.</a> Curr Biol. 18, 270-275 (2008).</li><li>Reiser, M. B., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modular+display+system+for+insect+behavioral+neuroscience.">A modular display system for insect behavioral neuroscience.</a> J Neurosci Methods. 167, 127-139 (2008).</li><li>G&#246;tz, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Course-control,+metabolism+and+wing+interference+during+ultralong+tethered+flight+in+Drosophila+melanogaster.">Course-control, metabolism and wing interference during ultralong tethered flight in Drosophila melanogaster.</a> J. Exp. Biol. 128, 35-46 (1987).</li></ol>

このシステムのための画像取得には、MATLABで書かれた安価なFireWireのボードカメラとソフトウェアを利用しています。明確な画像は、正確なデータのトレースを取得するために重要です。上記の戦略は、このシステムで画像をクリーンアップするのに便利です。このシステムの他のビデオトラッキングの手順は1記載されている。さらに、このシステムは、簡単にリアルタイムのビデ?...

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visually mediated odor tracking during flight in drosophila

フライング昆虫は、風の流れでその見出しを安定させるために視覚的な手がかりを使用してください。多くの動物は、さらに風で運ばれた匂いを追跡。このように、風上の視覚的な安定は、臭気の追跡に直接補助追跡。しかし、嗅覚の信号は、直接風の手がかりから独立して視覚的な追跡の動作に影響を与えるのですか？さらに、嗅覚の分子遺伝学と神経生理学の最近の進歩は、（例えば）遺伝的に特定の嗅覚のアクティベーション回路を不活性化の行動の影響を評価する新たな定量的な行動解析を動機としている。我々は元々狭い層流臭プルームでアリーナを搭載することにより、ビジョンの実験のために考案された磁気テザーシステムを変更。ここでは、フライがつながれた後に実行し、磁気の分野でナビゲートすることができることができる実験に焦点を当てる。我々は、安定した臭いの追跡を判断する方法、身体の角度を測定するために最適化されたビデオ画像を取得する方法を示して、そして我々は、臭気の追跡を視覚的合図の影響を調べるために2つの実験を示しています。

Watch this Scientific Journal Video about Visually Mediated Odor Tracking During Flight in Drosophila at JoVE.com

Visually Mediated Odor Tracking During Flight in Drosophila

ここでは、嗅覚磁気テザー（OMT）装置のために取得されたビデオ画像を最適化する方法について説明します。我々はまたvisuo -嗅覚融合を研究するための2つのサンプルの実験プロトコルを記述する。

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 ...

visually-mediated-odor-tracking-during-flight-in-drosophila

Research

JoVE Journal

Biology

9.8K ビュー.  University of California, Los Angeles. ここでは、嗅覚磁気テザー（OMT）装置のために取得されたビデオ画像を最適化する方法について説明します。我々はまたvisuo -嗅覚融合を研究するための2つのサンプルの実験プロトコルを記述する。

ビデオ: Visually Mediated Odor Tracking During Flight in Drosophila

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

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. ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

アダルトキイロショウジョウバエのロコモーションの高解像度のビデオトラッキング

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

シナプス電位のChannelrhodopsin2仲介刺激でショウジョウバエ神経筋接合部

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

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 ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

キイロショウジョウバエ成虫の間接飛翔筋の光学断面筋決定

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...