この作品は、NIAからYW.CF（AG21068、AG31086）への補助金によって支えられている。

1。インスリン溶液の調製<ol><li>は0.01 mg / mlの濃度を達成するためにPBSでインスリンを溶解することにより新鮮なウシのインスリン溶液を調製します。インスリン/ PBSおよび制御PBSの両方のソリューションは、注射の手順では氷上で保存する必要があります。これらのソリューションは、0.5％（v / v）のFD＆Cブルーなしで準備する必要があります。 1食品着色料。 </li></ol> 2。針の準備とセットアップインジェクション<ol><li>マイクロピペットpuller.Theを使用してガラスキャピラリーの針を準備し、以下の引き手の設定は、注射のための十分な品質の針の生成：熱、345、プル、210、速度、100;時間、200（100ミリ秒）。たて針は、キムワイプの組織を通じて、先端を押すことによって平滑化されている必要があります。この鈍化過程では、細長い高抵抗チップを削除し、より大きな細孔径を持つstouterのヒントを生成します。 </li><li>単独でインシュリン/ PBS溶液またはPBSを含むマイクロ遠心チューブにチップアッププレース鈍い針。各針のバック充填でアクションの結果をキャピラリー。ない破片や気泡が先端に表示されていないことを保証するために実体顕微鏡下で針の先端を観察。きれいに記入していない任意の針を捨てる。 </li><li>針の先端が実体顕微鏡を通して見えるようにマイクロマニピュレータを手動マイクロインジェクターのニードルホルダーとの位置ニードルホルダーに充填して針を挿入します。微量注入器の注射器のプランジャーに接続されている手動マイクロインジェクターのマイクロメーターのノブを回して、針の流体列に正圧を適用します。 </li><li>その流体の変位のための十分な圧力が針の先端にキムワイプを触れると流体の流れを確認することで適用されていることを確認します。 </li><li>いったん針が明確なテープ付き注射、貼付ニードルシャフトにキャリブレーションチャートの準備、および20倍の倍率下実体顕微鏡を介して焦点に針の先端を持参されています。 </li></ol> 3。準備を飛ぶ<ol><li> 10日齢の雌ハエは、注射の実験に使用されています。羽化後24時間以内にハエを集める。男性と場所雌ハエ標準出力または治療の食事療法を含むバイアルにガスのパッド上に加湿CO 2、ソートでハエを麻酔。 10日間実験飼料のメスのハエを維持する。 </li><li>実験飼料の上で羽化し、分離後10日目に、転送が2％寒天5mlのプラグを含むバイアルに彼らの食糧を含むバイアルの便を運行する航空会社と12〜16時間にわたってそれらを飢えさせる</li><li>インスリン注射前1時間、10％グルコース浸したフィルターを含むバイアルに飢えたハエを移す。 </li><li>簡潔にグルコースの給餌後に加湿CO 2でハエを麻酔してから、冷たい氷の上に固定化する。 </li></ol> 4。注射の手順<ol><li>優しく細かいピンセットを持つコールド固定化フライをつかみ、先端はフライの胸部の左側の前方領域に隣接するようにそれは針の先端に近いホールド。実体顕微鏡下で焦点に針の先端とフライの胸部の両方をもたらす。 </li><li>針の先端がフライの胸部（図1）の左前楯板領域の隆起の中心に触れるようにゆっくりと針の先端に向かって飛んで移動する。フライが適切な方向と針の先端で整列されると、針が前楯板の中心をimpalesように針の上にフライを進めていく。 </li><li>流体の0.1μlのハエに注入されるまで、マイクロインジェクターのマイクロマニピュレータのノブとシリンジのプランジャーを進めることによって、必要に応じて正圧を適用します。ハエの血体腔への流体の流れは、前胸部の左側に青い色を与えるだろう。時折一つは、任意の針の先端の閉塞を取り除くと流体の流れを可能にするために数回を収納して進める必要があります。 </li><li>注入されたハエは、血リンパの抽出の前に指定された時点で2％寒天バイアルに回復することができます。 </li></ol> 5。血リンパコレクション<ol><li>簡単に言うと、目的のポスト噴射の時点と位置で加湿CO 2でハエを麻酔背側を下にCO 2パッドの表面に貼付両面テープ上の各フライ。整列頭部カプセルと偶数行でハエを手配。一つは、テープの接着剤に羽を押すと固定化を確保するためにトリミングされた木製のアプリケータースティックを使用することができます。 </li><li>頭部カプセルの前面にステレオ顕微鏡を通して見えるように一度ハエが、テープに固定細かい鉗子でそれぞれのハエの口吻を持ち、ゆっくりとフライの腹側と後、後部領域に向かってそれを引いている。 </li><li>もう一方の手を持つとの位置に口吻を保持しながら、穿刺だけでシャープなタングステン針（図2）を使用してptilinal縫合上記頭部カプセルの中心地。一つは、あまりにも遠く、それは頭部のカプセルの他の側面に針の先端をパンチするのは簡単ですし、続いてcを失うように針を挿入しないように注意する必要があります血リンパの流れのontrol。穿刺は全血リンパを収集する前に注射群で飛ぶ。 </li><li>頭部カプセルが破裂されると、そっとその場の腹部に圧力を適用するためにトリミングされた木製のアプリケータースティックを使用してください。それはあなたが圧力を適用すると、その腹側に置いたようにフライをロールオーバーすると便利かもしれません。この手順では、頭部カプセル穿刺部位から血リンパの液滴が生成されます。 </li><li>頭部カプセル穿刺部位から出てきた体液の液滴に1μlのマイクロキャピラリーチューブの一方の端をタッチしてください。血リンパは、毛細管現象を介してチューブを入力します。卒業ボリュームのチャートに対して、マイクロキャピラリーチューブ内の流体の列をチェックして収集した血リンパの量を決定し、記録します。 </li></ol> 6。体液のグルコース定量<ol><li>インフィニティグルコース液100μlを含むマイクロプレートウェルに体液のサンプルを取り出します。血リンパのサンプルをロードしながら氷の上でプレートを保管してください。 </li><li> 50mmの25mmの、12.5mmの、6.25mMと3.125mMで別々に1μlのグルコースストック溶液のそれぞれをロードすることによって標準曲線を確立する。 </li><li> 37サンプルをインキュベート° Cで1​​0分間。 </li><li> 340nmで吸光度を検出する。 </li><li>血リンパのボリュームのアカウントが収集され、既知のグルコース濃度の標準曲線に基づいて、サンプルのグルコース濃度を決定する。 </li></ol> 7。代表的な結果典型的なインスリン耐性の応答はグルコースの循環レベルの低下は15分後の注射を検出されるインスリン注入ハエで検出される。対照的に、そのような応答は、PBSを注入ハエ（図3）には見られません。末梢グルコース処理のこの応答は30分後に注射するインスリン注入のハエで続行されます。我々は定期的に各注射群では4〜5ハエごとに血リンパの0.2から0.5μlを抽出する。三注入グループは各実験に含まれています。 <img src="/files/ftp_upload/2722/2722fig1.jpg" alt="図1"/> 図1針の挿入部位（Demerec、1950年から変更された） 中7件を表示ショウジョウバエの胸部の左側。胸部の左側の前部、背部上の前楯板の中心を通って針を挿入します。 <img src="/files/ftp_upload/2722/2722fig2.jpg" alt="図2"/> 図2血リンパの抽出のための穿刺の場所（Demerec、1950年から変更された） 中7件を表示ショウジョウバエの頭部の正面図。穿刺だけptilinal縫合上記頭部カプセルの中央に細かく削ったタングステンプローブを備えたヘッドカプセル。 <img src="/files/ftp_upload/2722/2722fig3.jpg" alt="図3"/> 図3：コントロールの成人で検出された典型的なインスリン負荷応答が飛ぶ。コントロールワット1118ハエは、ウシインスリン（PBS中の1 NG）またはPBSのみを注射した。複製グループのハエは、0、15または30分間回復させたと循環グルコースレベルを測定した。

<ol>
	<li>Rulifson, E. J., Kim, S. K., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+insulin-producing+neurons+in+flies:+growth+and+diabetic+phenotypes.">Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes.</a> Science. 296, 1118-1118 (2002).</li><li>Kim, S. K., Rulifson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+mechanisms+of+glucose+sensing+and+regulation+by+Drosophila+corpora+cardiaca+cells.">Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells.</a> Nature. 431, 316-316 (2004).</li><li>Broughton, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longer+lifespan,+altered+metabolism,+and+stress+resistance+in+Drosophila+from+ablation+of+cells+making+insulin-like+ligands.">Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands.</a> Proc Natl Acad Sci U S A. 102, 3105-3105 (2005).</li><li>Haselton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+ablation+of+adult+Drosophila+insulin-producing+neurons+modulates+glucose+homeostasis+and+extends+life+span+without+insulin+resistance.">Partial ablation of adult Drosophila insulin-producing neurons modulates glucose homeostasis and extends life span without insulin resistance.</a> Cell Cycle. 9, 3063-3063 (2010).</li><li>Haselton, A. T., Fridell, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+Drosophila+melanogaster+as+a+model+for+the+study+of+glucose+homeostasis.">Adult Drosophila melanogaster as a model for the study of glucose homeostasis.</a> Aging (Albany NY). 2, 523-523 (2010).</li><li>Belgacem, Y. H., Martin, 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=Disruption+of+insulin+pathways+alters+trehalose+level+and+abolishes+sexual+dimorphism+in+locomotor+activity+in+Drosophila.">Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila.</a> J Neurobiol. 66, 19-19 (2006).</li><li>Demerec, 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> Biology of Drosophila. , (1950).</li></ol>

利害の衝突は宣言されません。

このレポートに記載されている方法は、 ショウジョウバエの血リンパの組成の検出可能な変化をもたらす生理学的プロセスを調査するあらゆる研究に有用である可能性がある。この方法で注入し、血リンパのコレクションを組み合わせることで、それは特定の実験的治療または操作の直接の生理学的に関連する影響を把握することが可能です。断頭2を含む以前の技術よりも体...

<table border="1"><tbody><tr><th>試薬/装置の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>ウシインスリン</td><td>シグマ</td><td> I5500 </td></tr><tr><td>無限のグルコース試薬</td><td>サーモエレクトロン株式会社</td><td> TR1541 </td></tr><tr><td>手動マイクロインジェクター</td><td>サターのインストゥルメント</td><td></td></tr><tr><td> P - 87 Flamming /ブラウンマイクロピペットプラー</td><td>サターのインストゥルメント</td><td></td></tr><tr><td>シングルバレルホウケイ酸ガラスキャピラリー</td><td> AMシステム</td><td> 626000 </td></tr><tr><td> FD＆C青色1号</td><td>マコーミックアンドカンパニー</td><td></td></tr><tr><td> 1μlのマイクロキャピラリーチューブ</td><td>ドラモンド</td><td></td></tr><tr><td>三軸の手動マイクロマニピュレーターとベース</td><td>世界の精密機器</td><td></td></tr></tbody></table>

insulin injection and hemolymph extraction to measure insulin sensitivity in adult drosophila melanogaster

保存された栄養感知のメカニズムが発達幼虫期の1,2時の血糖値の恒常性を維持するために、それぞれのペプチドは哺乳類のインスリンとグルカゴンに似た哺乳類やショウジョウバエ、機能間に存在する。主に有糸分裂後の成人のハエの研究は、インスリン様ペプチドの遺伝子除去（ILP）産生細胞（IPC）を3の結果としてグルコースの恒常性の摂動を明らかにした。したがって、成人ミバエは、II型糖尿病を含む代謝性疾患に適した遺伝的モデルシステムとして有望視されて保持する。さらにショウジョウバエのシステムを開発するには、哺乳動物における耐糖能とインスリン感受性を測定するために使用される同等の生理的アッセイを確立する必要があります。この目的のために、我々は最近、成人のハエにおける経口ブドウ糖負荷応答を測定するための新たな手順を記載していると4,5グルコース恒常性を維持するに成人IPCの重要性を示した。ここで、我々は、インスリンの注射6前述の手順を変更し、成体のハエで、末梢インスリン感受性を測定するために新たな血リンパの抽出法とそれを組み合わせている。一意に、我々のプロトコルは、インスリン注射、インスリンシグナル伝達変異体と大人のハエの耐糖能とインスリン感受性に影響を与える潜在的な介入を特徴づけることが可能になる方法論に応じて末梢グルコース負荷を処分する大人ハエの能力の直接的な生理学的な測定が可能です。

Watch this Scientific Journal Video about Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster at JoVE.com

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

ショウジョウバエに見られる保存されたインスリンのシグナル伝達経路<em&gt;キイロショウジョウバエ</em&gt;この生物II型糖尿病を含む代謝性疾患をモデル化するための潜在的なツールとなっています。そのためには、効果的に成人ハエの末梢グルコース処理の全身のインスリン作用を測定するための生理学的アッセイを確立することが重要です。

インスリン注射と成人のインスリン感受性を測定する血リンパの抽出キイロショウジョウバエ</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

insulin-injection-hemolymph-extraction-to-measure-insulin-sensitivity

Research

JoVE Journal

Biology

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

19.6K ビュー.  State University of New York. ショウジョウバエに見られる保存されたインスリンのシグナル伝達経路キイロショウジョウバエこの生物II型糖尿病を含む代謝性疾患をモデル化するための潜在的なツールとなっています。そのためには、効果的に成人ハエの末梢グルコース処理の全身のインスリン作用を測定するための生理学的アッセイを確立することが重要です。

ビデオ: Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

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 ...

生物学

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 ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

インスリン発現コロニーを形成する前駆細胞の定量アッセイ

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

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 ...

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The Drosophila&#160;melanogaster Malpighian (renal) tubule offers an unparalleled opportunity to study the molecular mechanisms of epithelial ion transport, due to the powerful genetics of this organism and the accessibility of its renal tubules to physiological study. Here, we describe the use of the Ramsay assay to measure fluid secretion rates from isolated fly renal tubules, with the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid. This assay allows study of transepithelial fluid and ion fluxes of ~20 tubules at a time, without the need to transfer the secreted fluid to a separate apparatus to measure ion concentrations. Genetically distinct tubules can be analyzed to assess the role of specific genes in transport processes. Additionally, the bathing saline can be modified to examine the effects of its chemical characteristics, or drugs or hormones added. In summary, this technique allows the molecular characterization of basic mechanisms of epithelial ion transport in the Drosophila tubule, as well as regulation of these transport mechanisms.

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

This protocol describes the use of the Ramsay assay to measure fluid secretion rates from isolated Malpighian (renal) tubules from Drosophila melanogaster. In addition, the use of ion-specific electrodes to measure sodium and potassium concentrations in the secreted fluid, allowing calculation of transepithelial ion flux, is described.

で液分泌とイオンフラックス速度を測定するためのラムゼイアッセイの使用<em&gt;キイロショウジョウバエ</em&gt;マルピーギ管

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

インスリン刺激に対するグルコース取り込み測定と応答<em&gt;インビトロ</em&gt;培養ヒト初代筋管

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Confocal Imaging of Neuropeptide Y-pHluorin: A Technique to Visualize Insulin Granule Exocytosis in Intact Murine and Human Islets

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study insulin granule exocytosis either use electrophysiology or microscopy coupled to the expression of fluorescent reporters. However most of these techniques have been optimized for clonal cell lines or require dissociating pancreatic islets. In contrast, the method presented here allows for real time visualization of insulin granule exocytosis in intact pancreatic islets. In this protocol, we first describe the viral infection of isolated pancreatic islets with adenovirus that encodes a pH-sensitive green fluorescent protein (GFP), pHluorin, coupled to neuropeptide Y (NPY). Second, we describe the confocal imaging of islets five days after viral infection and how to monitor the insulin granule secretion. Briefly, the infected islets are placed on a coverslip on an imaging chamber and imaged under an upright laser-scanning confocal microscope while being continuously perfused with extracellular solution containing various stimuli. Confocal images spanning 50 &#181;m of the islet are acquired as time-lapse recordings using a fast-resonant scanner. The fusion of insulin granules with the plasma membrane can be followed over time. This procedure also allows for testing a battery of stimuli in a single experiment, is compatible with both mouse and human islets, and can be combined with various dyes for functional imaging (e.g., membrane potential or cytosolic calcium dyes).

We describe a protocol for visualization of insulin exocytosis in intact islets using pHluorin, a pH-sensitive green fluorescent protein. Isolated islets are infected with adenovirus encoding pHluorin coupled to the vesicle cargo neuropeptide Y. This allows for the detection of insulin granule fusion events by confocal microscopy.

共焦点イメージングの神経ペプチド Y-pHluorin: そのままのマウスと人間のランゲルハンス島におけるインスリン開口分泌の可視化技術

Insulin secretion plays a central role in glucose homeostasis under normal physiological conditions as well as in disease. Current approaches to study ...

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 ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

口腔の細菌感染とキイロショウジョウバエの開口

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

ライブショウジョウバエメラノガスター卵室における内因性mRNAの可視化と追跡

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...