著者は、知的支援、物質的な支援、および株式のために明千葉を認識したい。コメントのジュリアDallmanに感謝します。

1。作業を飛ばす<ol><li>室温10で、標準的なコーンミール寒天·糖蜜·酵母媒体上のハエを維持する。 </li><li>十字架は、羽化の6時間以内で処女を分離します。希望の遺伝子型の男性に横切った後、変更は3〜4日ごとに新しいバイアルに飛ぶ。 </li></ol>注：これらの実験では、Gal4の線A9がウィングに導入遺伝子の発現を駆動するために使用した。フライ株はブルーミントンストックセンターから入手することができます。これらの実験で使用した株はA9-GAL4（のBL＃8761）、His2Av-GFP（のBL＃5941）、SCO / CYO HsCre（のBL＃1092）、UAS-ChRFP-タブ（のBL＃25773）、lollibow 11が含まれています。 2。蛹の選択と取り付け<ol><li>ステージ蛹のどちらか白prepupae（WPP）としてそれらを収集し、それらが適切な年齢に達するまで、25℃でそれらを維持するか、形態学的基準12を使用します。 </li><li>細胞分裂、最近のヘッド外転を受けたセレクト蛹を観察する。この時点からわずか羽化直前まで（&gt; 96時間の時点で）、蛹は不動長時間タイムラプス観察を許可している。 </li><li>最初の水は絵筆にドラッグつついそっとして接着剤を緩めて、できるように分を待って、水で湿らせた絵筆でタッチしてバイアルから蛹を削除します。 </li><li>優しく唾液腺接着剤、食品粒子を除去し、水に絵筆でそれらをつついで選択された蛹を洗う。 </li><li>カバースリップ底（番号1.5カバーグラス）で25ミリメートルペトリ皿にきれいな蛹を転送します。 </li><li>目的の組織をカバーガラスに最も近くなるようサポートなどの粘土や歯科用ワックスのどちらかの薄いストリップを使用して、蛹をマウントします。 注意：ここに記載された研究では、蛹の翼、脚、腹部histoblastsまたは背側胸背板が観察されている。実際には、蛹のケースの表面の20ミクロン内の任意の組織が観察される。 </li><li>オリエント蛹慎重になるようTISSU関心のあるeは、カバーガラス表面に平行である。 </li><li>蛹がマウントされると、蛹とカバーガラスの間の空間にチオジエチレングリコール（TDG）の薄層を転送するために絵筆を使用しています。 </li></ol>注：TDGは、表面散乱を低減するオイルの屈折率と一致し、酸素が ​​組織13に浸透することを可能にする。それらが細胞の挙動を迅速に停止を引き起こし、酸素の組織を奪う傾向があるので、油の使用は、お勧めしません。 3。イメージング注：画像は、共焦点顕微鏡は、共焦点性が蛹ケースの強烈な照明によって生じる隠蔽の背景のほとんどが削除された必須である可能性が高い。 <ol><li>蛹の開発と生存率に照明の影響を最小限に抑えるために、共焦点の設定を調整します。そのためには、励起の強度と収集の感度のバランスを取る。イメージングWIの一般的な設定ライカSP5番目の120μmの共振スキャナ（8,000 Hz）で、2％（20％の電力の10％の透過率）に設定されたレーザパワー、ピンホールのセットを使用し、ラインが8に設定されアベレージ。設定は、実験条件に依存して変化する。 </li></ol>注：これらは、焦点深度を制限するのに細かいスケールを解決するには40Xと63X油浸レンズ（0.1ミリメートル作動距離）を備えています、また成功し、この方法で使用されてきた。 4。分析フレームを分析するために、Zスタックや映画は、見ての測定、およびプレゼンテーション14用のファイルを修正するための効果的なツールを持っているフィジー、にデータファイルをインポートします。例えば、有糸分裂を完了する時間は、前期から終期までの細胞を見ながらフレームをステップサンプリング間隔および多次元画像ブラウザを使用して測定した。 x軸、y軸、およびz寸法は、測定ツールを用いて測定することができる。ソフトウェアとその使用方法の詳細は、参照：http://fiji.sc/Fijiを。

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著者らは、開示することは何もありません。

、指標を可視化し、分裂細胞の特徴を定量化するために、His2AvGFP発現している細胞の共焦点解析により、ショウジョウバエの生活蛹の翼で有糸分裂を観察するための簡単な準備の開発を必要とした。このメソッドは、蛹の翅における細胞周期は、彼らが有糸分裂に入る上皮の頂端面への核の移行に多列上皮で細胞周期に強い類似性を有することを文書化するために使用された。終期に...

<html>酢のフライ、 キイロショウジョウバエは 、生物学の多くの側面を研究するための十分に確立されたモデルである。 ショウジョウバエの研究は表情、ノックダウン突然変異を含む遺伝子操作の洗練されたフォームを可能にする遺伝的な実験の豊かな歴史を持っています。蛍光タンパク質標識の出現により、このレパートリーは動物生体内の細胞およびタンパク質の研究を含むように拡大している。ハエ胚は、 生体内で 1-3 の深い、高分解能イメージングを可能に小さく、光学的に透明であるように、このような研究のための優れたシステムです。ハエの発達の他の段階はanaesthetization 4、切開および短期培養5,6、又は7,8イメージングのためのクチクラにおけるウィンドウの作成 ​​を必要とする、より少なく扱いやすいことが証明されている。これらの操作は、通常、長期的には、動物の発達を損なうか、短時間に画像を制限する方法で動物に影響を与える。 ほとんどの胚細胞サイクルが切り捨てられているので、細胞周期調節因子に似ている遺伝子における新規の変異を研究するENT &quot;&gt;、それが細胞周期のタイミングと忠実度を研究するための適切な準備を見つけることが不可欠であった（SMまたはS-G2-M）と研究対象の変異体は後の段階までの欠陥を示していない、それは蛹組織における細胞周期を観察することが重要だった。蛹の上皮細胞は、この段階でのより一般的なG1-S-G2-M細胞周期や蛹を持っている筋肉の動き9できない。操作のための最初の出発点はHistone2AV-GFPを発現する完全な全体蛹が含まれていました。蛹ケースの見かけの不透明度にもかかわらず、この完全な準備がin vivoイメージングの長期のための優秀なことが証明された。この手法は単純である学部の研究者が日常的にショウジョウバエの細胞と発生生物学の側面を研究するためにそれを使用して、まだ解決は、マイクロメートルスケールの特徴の識別を可能にするために十分な罰金であることを十分に。この方法では、時間、分、秒を超える事象の観察は、単に時系列パラメータを調整することによって可能である。青色、緑色、黄色、橙色、および赤色蛍光タンパク質、又はこれらの組み合わせを用いて動画を、なされている。重要なことに、もし注意があっても、長期的画像化は、動物の発症または生存率には影響がなく、レーザ強度​​を最小化するために取られる。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly stuff fly pad</td>
			<td style="width:71px;">Genesee scientific</td>
			<td style="width:119px;">59-114</td>
			<td style="width:167px;">for fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CO2 gas</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;">CD50</td>
			<td>For fly anesthetization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regulator</td>
			<td style="width:71px;">Airgas south</td>
			<td style="width:119px;"> </td>
			<td>CO2 regulator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fly vials</td>
			<td style="width:71px;">Genesee scientific</td>
			<td>32-113RLBF</td>
			<td>Fly culture</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Drosophila lines:A9-Gal4 (Bl#8761), His2Av-GFP (Bl#5941), Sco/CyO HsCre (Bl#1092), UAS-ChRFP-Tub (Bl#25773)</td>
			<td style="width:71px;">Bloomington Stock center</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass bottom dishes #1 1/2</td>
			<td style="width:71px;">WillCo Wells BV</td>
			<td style="width:119px;"> </td>
			<td>For microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thiodiethylene Glycol</td>
			<td style="width:71px;">Fluka</td>
			<td align="right" style="width:119px;">88559</td>
			<td>mountant</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Modeling clay</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>Support to position pupae against</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paintbrushes</td>
			<td style="width:71px;">art supply store</td>
			<td style="width:119px;"> </td>
			<td>To manipulate flies</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fine Forceps, Inox #5</td>
			<td style="width:71px;">Fine science tools</td>
			<td style="width:119px;">11252-20</td>
			<td>Dumont #5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">computer</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>8 Gb RAM for image/movie analysis</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">Fiji software</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;">Free ware http://fiji.sc/Fiji</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td>Any fast scanning confocal should be sufficient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">20x dry, and 40x or 63x oil immersion lenses</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For imaging tissue, cellular and subcellular features</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil (non fluorescent)</td>
			<td style="width:71px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:71px;">any</td>
			<td style="width:119px;"> </td>
			<td>For fly manipulation</td>
		</tr>
	</tbody>
</table>

imaging through the pupal case of drosophila melanogaster

細胞および発生生物学のモデルとしてショウジョウバエの長年の使用によりツールの配列が得られている。一緒に、これらの技術は、方法論的な様々な角度からの細胞と発生生物学の分析を可能にした。ライブイメージングは​​、細胞分裂または細胞運動性などの動的な細胞プロセスを観察するための新たな方法である。特徴付けられていない推定上の細胞周期タンパク質に変異を有する単離された、ライブイメージングを用いてその場で有糸分裂を観察することが必須となった。 ショウジョウバエで最もライブイメージング研究は、それらのサイズが小さく、光学的透明性の操作や観察にアクセス可能な胚の段階に焦点を当てている。それはギャップ相の一方または両方を欠くという点でしかし、これらの段階において細胞周期は珍しい。対照的に、 ショウジョウバエの蛹の翼の細胞は、典型的な細胞周期を有し、蛹開発の約20時間に及ぶ急速な有糸分裂の周期を経る。それは私には簡単ですdentifyとその場で有糸分裂をキャッチし、適切なステージの蛹を分離します。そのまま蛹をマウントする実験は、細胞や動物の生存能力への影響を最小限に抑えながら、数時間のために実行できるように、撮像中に扱いやすと耐久性の最適な組み合わせを提供した。この方法は、小さな、あるいはより小さな、染色体を飛ぶような特徴を観察することができます。顕微鏡の設定や隣接するセルや、チューブリンなどの蛍光標識されたタンパク質の膜動態を可視化するための準備の設置、許可された拡張機能の詳細を調整する。この方法は、すべてのテストされた蛍光タンパク質のために働くと時間スケールの様々な経由でサブミクロンスケールの特徴を捉えることができます。従来の共焦点顕微鏡で蛹の外側を20μmに限定しながら、 生体内での蛹の組織で、タンパク質や細胞動態を観察するためのこのアプローチは、これらの組織中の細胞および発生生物学の研究に一般的に有用である可能性がある。

このような現像ショウジョウバエの眼、又は現像脊椎動物の中枢神経系の心室層として多列上皮における細胞は、核の運動を起こす、細胞周期に合わせて、interkinetic核移行と呼ばれる。核が基底面またはその近くで、核は頂端面15,16に達したとき、細胞が有糸分裂を入力すると、DNAの複製が発生します。蛹の翼細胞は頭部外転後の最初の数時間の間に急速に分裂する単層上皮を...

Watch this Scientific Journal Video about Imaging Through the Pupal Case of Drosophila melanogaster at JoVE.com

Imaging Through the Pupal Case of Drosophila melanogaster

本論文では、直接蛹殻を介して画像細胞挙動への高速走査型共焦点顕微鏡を使用する方法を示します。そのまま蛹ケースを残すことによって、このメソッドを直接学ぶことは困難であるショウジョウバエの発生段階で動的な細胞プロセスの観察と測定を可能にします。

の蛹ケースを画像化<em&gt;キイロショウジョウバエ</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

imaging-through-the-pupal-case-of-drosophila-melanogaster

Research

JoVE Journal

Biology

Imaging Through the Pupal Case of Drosophila melanogaster

13.2K ビュー.  University of Miami. 本論文では、直接蛹殻を介して画像細胞挙動への高速走査型共焦点顕微鏡を使用する方法を示します。そのまま蛹ケースを残すことによって、このメソッドを直接学ぶことは困難であるショウジョウバエの発生段階で動的な細胞プロセスの観察と測定を可能にします。 

ビデオ: Imaging Through the Pupal Case of Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

のライブイメージングキイロショウジョウバエ胚性血球移行

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

の直立イメージングショウジョウバエ胚

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

大人からOenocytesの解剖キイロショウジョウバエ</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

の分離キイロショウジョウバエ精巣

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

ショウジョウバエの蛹の腹部の免疫組織化学

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖、イメージング、および薬理学的治療： ショウジョウバエ蛹精巣や独身男性生殖ライン嚢胞のex vivo培養

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖と実装<em&gt;ショウジョウバエ</em&gt;蛹アイディスク

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

のメンテナンス<em&gt;キイロショウジョウバエ</em&gt;人口ケージ

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...