This work was supported by Adelphi University faculty development grant and grant R15GM113156 from NIGMS awarded to EVC, grant R01GM067862 from NIGMS and grant R01AG027849 from NIA awarded to DMR.

1.試薬およびソリューション<ol><li>フライ食品やメディアの準備<ol><li>熱フライ成分90℃に設定したホットプレートで水にw / vの0.79パーセント寒天11％の砂糖、2％自己消化酵母、5.2％のコーンミール、。均質やや密な混合物が得られるまで定期的に攪拌します。各食品を飛ぶmlの5と100バイアルの合計フライ食品の550ミリリットルの最終容量を準備します。 </li><li> 、加熱源から削除食品がダウン80℃に冷却するまで、時々攪拌し、95％エタノールに溶解した0.2％tegosept-4-ヒドロキシ安息香酸メチルを加えます。 </li><li> 2等量の食品を分割します。他のボリュームへの1つのボリュームとエタノール1.1 mlにエタノールに溶解したラパマイシンの50mMの1.1ミリリットルを追加します。ラパマイシンおよび食品へのエタノール溶液を攪拌します。温度が薬を追加する前に、50℃に低下していることを確認してください。 </li></ol></li><li>分離バッファ<ol><li>単離緩衝液[225mMのマンニトール、75mMのスクロース、10mMの3-（N &lt;500mlのを準備します/ em&gt;のモルホリノ）プロパンスルホン酸（MOPS）および1mMエチレンジアミン四酢酸（EDTA）、2.5 mg / mlのウシ血清アルブミン（BSA）]水に。 </li><li>水酸化ナトリウムを用いてpHを7.2に調整します。初期のpHが酸性になります。 </li><li>ソリューションを殺菌し、4℃でそれを保存するために、0.22μmの細孔径を有する滅菌フィルターの保存瓶を使用してください。 </li></ol></li><li>洗浄バッファー<ol><li>水で洗浄緩衝液[225mMのマンニトール、75mMのスクロース、10mMの塩化カリウム、10mMのトリスHClおよび5mMのKH 2 PO 4]の500ミリリットルを準備します。 </li><li>水酸化ナトリウムを用いてpHを7.2に調整します。初期のpHが酸性になります。 </li><li>ソリューションを殺菌し、4℃でそれを保存するために、0.22μmの細孔径を有する滅菌フィルターの保存瓶を使用してください。 </li></ol></li></ol> ショウジョウバエ株の2飼育<ol><li> 、開発中の幼虫密度を制御培養瓶に両親の25組を配置し、それらが48時間の卵を産むことを可能にします。 </li> &lt;LI&gt;卵の密度を調整するために48時間後の親オブジェクトを削除します。 </li><li>繰り返しは、新興のF1子孫を使用して、2.1と2.2の手順ので、この濃度制御の二世代が達成されます。 </li><li>大人を収集するには、二酸化炭素（CO 2）と性別によって別々に使用してハエを麻酔。 <ol><li> 5 PSIの連続的な流れで高圧のタンクから、純粋なCO 2を配信するために単段レギュレータを使用してください。静電気を避けるために、水を用いてフラスコをフィルタリング500ml中にCO 2を触媒するためにプラスチックチューブを使用します。 </li><li>フラスコを密封するために一つの穴にゴム栓を使用してください。 CO 2パッドにフラスコの側方開口部を接続するためのプラスチック製のチューブを使用してください。 </li><li> 10〜15分を超えないためのパッドでハエを置きます。実験条件でそれらを配置する前に24時間、麻酔から回復できるようにします。 </li></ol></li><li>ミトコンドリアの濃縮された画分のための十分な代謝物を生成するには、サンプルあたり300ハエを使用しています。ここでは、女性を使用していますが、genderは他の実験では異なる場合があります。自家製1Lの人口統計ケージに150ハエを転送します。バイアル中のフライ食品の5ミリリットルへのアクセスを許可します。 </li><li>別の1リットルケージに残りの150ハエを転送します。 150以上のハエでケージを人口過剰にするしないでください。 </li><li>実験条件ごとに6つの複製サンプルを使用してください。全動物の抽出物は、動物全体の1サンプルを生成するために、より多くの代謝物を得ているので隔離し、転送50女性は1ケージに飛びます。ミトコンドリアの分離株については、実験条件ごとに6つの複製サンプルを使用しています。 </li><li> 12時間の光と12時間の暗のサイクルで25℃でのケージを置きます。 </li><li>食品の品質を維持するために、食品の5 mlのバイアル中で2〜3日ごとに新鮮な食品を提供します。 </li><li> 10日間の食品のハエを維持します。このステップは、ラパマイシン処理7のために必要とされます。他の治療法や条件が異なります。 </li></ol>ミトコンドリア画分の3の単離<ol><li> 1ケージ（150ハエ）からハエをダンプ冷却した単離緩衝液1mlを充填したガラス - テフロンダウンスホモジナイザー。 </li><li>ダウン15ストローク用乳棒アップとを移動させることにより、均質化します。無傷ミトコンドリアを維持するために氷の上にモルタルを続けます。 </li><li> 4℃で5分間、300×gで1.5mlチューブと遠心分離機にホモジネートを転送します。 </li><li>ミトコンドリアを濃縮するために4℃で10分間6000×gで上清および遠心してください。 </li><li>上清を細胞質画分を含んでいるので、上清を廃棄または別の実験のためにそれを使用しています。洗浄バッファー300μlの中にペレットを再懸濁。 </li><li>第2のかご用3.5 - を繰り返して、3.1を繰り返します。極低温マイクロチューブの両方でケージの再懸濁したペレットを組み合わせます。 </li><li> 4℃で10分間、6,000×gで遠心分離します。 </li><li>上清を捨て、液体窒素中でペレットをフラッシュ凍結。 </li><li> -80℃以下の温度で濃縮されたミトコンドリア画分を保管してください。 <ol><li>また、動物全体の事前のためのparation、極低温マイクロチューブに大人を配置します。 -80℃以下の温度で液体窒素や店舗成人のバイアルをフラッシュ凍結。 </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial-nuclear+epistasis+affects+fitness+within+species+but+does+not+contribute+to+fixed+incompatibilities+between+species+of+Drosophila.">Mitochondrial-nuclear epistasis affects fitness within species but does not contribute to fixed incompatibilities between species of Drosophila.</a> Evolution; international journal of organic evolution. 64 (12), 3364-3379 (2010).</li><li>Villa-Cuesta, E., Holmbeck, M. A., Rand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+increases+mitochondrial+efficiency+by+mtDNA-dependent+reprogramming+of+mitochondrial+metabolism+in+Drosophila.">Rapamycin increases mitochondrial efficiency by mtDNA-dependent reprogramming of mitochondrial metabolism in Drosophila.</a> Journal of cell science. 127 (Pt 10), 2282-2290 (2014).</li><li>Zhu, C. -. T., Ingelmo, P., Rand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G&#215;G&#215;E+for+Lifespan+in+Drosophila:+Mitochondrial,+Nuclear,+and+Dietary+Interactions+that+Modify+Longevity.">G&#215;G&#215;E for Lifespan in Drosophila: Mitochondrial, Nuclear, and Dietary Interactions that Modify Longevity.</a> PLoS genetics. 10 (5), e1004354 (2014).</li><li>Madala, N. E., Piater, L. A., Steenkamp, P. A., Dubery, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+statistical+models+of+metabolomic+data+reveals+different+metabolite+distribution+patterns+in+isonitrosoacetophenone-elicited+Nicotiana+tabacum+and+Sorghum+bicolor+cells.">Multivariate statistical models of metabolomic data reveals different metabolite distribution patterns in isonitrosoacetophenone-elicited Nicotiana tabacum and Sorghum bicolor cells.</a> SpringerPlus. 3, 254 (2014).</li><li>Hogeboom, G. H., Schneider, W. C., Pallade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+Studies+Of+Mammalian+Tissues+I+.+Isolation+Of+Intact+Mitochondria+From+Rat+Liver+Some+Biochemical+Properties+Of+Mitochondria+And+Submicroscopic+Particulate+Material.">Cytochemical Studies Of Mammalian Tissues I . 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著者らは、開示することは何もありません。

このプロトコルの中で最も重要なステップは次のとおりです：1）豊かな空間に十分なハエを飼育します。これは、150以上のハエそれぞれと人口統計ケージを人口過剰にするしないことが非常に重要です。 2）食品の競争と栄養ストレスを避けるために頻繁にケージの食品を変更します。 3）ミトコンドリア画分の単離の間に整合性を確保するために4℃ですべてのサンプルを維持します。また?...

この手順の目標は、 キイロショウジョウバエを用いたメタボロミクス研究のために十分なミトコンドリア代謝物を得濃縮されたミトコンドリア画分を開発することです。我々の経験では、全細胞抽出法を用いたメタボロミクス解析は、 ショウジョウバエの微妙なミトコンドリアの代謝産物の変化を検出することができません。しかし、従来メタボロミクス解析にミトコンドリア分画、ミトコンドリア代謝産物シフトを同定するための感度を増加させます。 ミトコンドリアは、細胞が正常な機能1に必要なエネルギーの90％を提供する責任細胞小器官です。近年では、ミトコンドリアが単にアデノシン三リン酸（ATP）を産生するよりも細胞および生物機能においてはるかにダイナミックな役割を果たし、現在は代謝恒常性2,3の調節のためのハブとして認識されていることが認識されています。ミトコンドリアは、DISいる内共生プロセスの結果であります着色した微生物の系統は、15億年前の4〜合併しました。ミトコンドリアは、真の細胞小器官へと進化したように、内部共生からの遺伝子は、新興核ゲノムに組み込まれました。 37遺伝子は酸化的リン酸化5の酵素複合体のサブユニットであるミトコンドリアのタンパク質をコードする13の、ミトコンドリアDNAに残したままの動物では、今日、約1,500ミトコンドリアタンパク質は、核コードです。ミトコンドリアおよび核コンパートメント間の調整は、適切なミトコンドリア機能を維持するために必要とされます。 ここに記載された方法を用いて、我々は、ミトコンドリアと核ゲノムとの間の調整の操作から生じるショウジョウバエにおけるミトコンドリアの代謝変化を検出することができました。私たちは、その姉妹種DからするのmtDNAにショウジョウバエのひずみを使用しましたシミュ単一D.上に配置しました核背景6 メラノ 。この「中断」mitonuclear遺伝子型はDの 「ネイティブ」、または共進化mitonuclear遺伝子型と比較しましたそのネイティブDに同じ核ゲノムを運ぶメラノミトコンドリアDNAをメラノ 。 D.ショウジョウバエとD.シミュの mtDNAsは〜100アミノ酸とmitonuclear通信7,8に影響を与える &gt; 500同義置換により異なります。私たちは、薬理学的なストレスに応答して代謝物のシフトを研究するために全体のフライを抽出し、ミトコンドリアの濃縮された抽出物を生成しました。ここでは、ミトコンドリアに富む画分を使用した場合、我々はDを運ぶネイティブ、共同発展の遺伝子型との間にミトコンドリアの代謝物の顕著なシフトを検出することを示しますショウジョウバエの mtDNAs と D を運ぶ破砕遺伝子型シミュのmtDNA。対照的に、これら2つの遺伝子型の間で代謝産物の変化は、全体のハエ抽出物を利用する通常の方法を用いて微妙です。したがって、この方法はmtDNAsがでミトコンドリアの変化を仲介する方法を理解するためのパスを提供しました異なる薬剤への応答。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.2% tegosept -methyl 4-hydroxybenzoate&#160;</td>
			<td>VWR</td>
			<td>AAA14289</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>792799</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(N-morpholino) propanesulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td>M1254&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>38057&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris HCL&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T-B7</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551139</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 pads to anesthetize flies</td>
			<td>Tritech Research</td>
			<td>MINJ-DROS-FP</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999RD32</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage lid&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999LRD</td>
			<td></td>
		</tr>
		<tr>
			<td>a glass-teflon dounce homogenizer&#160;</td>
			<td>Fisher Scientific</td>
			<td>NC9661231</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>rapamycin&#160;</td>
			<td>LC Laboratories&#160;</td>
			<td>R-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-porin</td>
			<td>MitoSciences</td>
			<td>MSA03</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-alpha tubulin</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>12G10</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#8482; BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 pad</td>
			<td>Tritech Research, Inc</td>
			<td>MINJ-DROS-FP</td>
			<td></td>
		</tr>
		<tr>
			<td>filter flask</td>
			<td>enasco</td>
			<td>SB08184M</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber stopper</td>
			<td>enasco</td>
			<td>S08512M</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of mitochondrial enriched fractions for metabolic analysis in drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

プロトコルを使用して上記で説明し、我々は発散mtDNAs 7にラパマイシン薬物の効果をテストするために、ミトコンドリアの濃縮された画分および全動物抽出物にメタボローム解析を行いました。私たちは、フライ食品で薬物を溶解することにより、ラパマイシンの200μMを配信しました。ハエは、10日間ラパマイシンに曝露しました。全フライ抽出物からの?...

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Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

代謝分析のためのミトコンドリア富化画分の調製で<em&gt;ショウジョウバエ</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

preparation-mitochondrial-enriched-fractions-for-metabolic-analysis

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Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K ビュー.  Adelphi University. Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster. 

ビデオ: Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

エバンスブルー色素でゼブラフィッシュ幼生骨格筋の整合性の分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

発生生物学

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

中規模の<em&gt;ショウジョウバエ</em&gt;プロテオーム実験のための胚抽出物

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

周辺嗅覚組織の開発を準備する分子とショウジョウバエの免疫組織化学的解析

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

ガスクロマトグラフィー-質量分析法 (GC/MS) のためのショウジョウバエ幼虫サンプル準備-メタボロミクスをベース

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

免疫組織化学、西部の分析、および RNA の隔離のためのショウジョウバエ蛹網膜の解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

ショウジョウバエ胚における筋収縮のライブイメージングと解析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

解凍、養殖、ショウジョウバエ細胞をトウキョウ

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

生きた無傷組織における細胞分裂解析のためのショウジョウバエ・ラーバルとプパル精巣の調製

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes&#39; zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve&#39;s regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (&#177; standard deviation) of 137,904 &#177; 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 &#177; 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 &#177; 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P &#60; 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P &lt; 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

牛卵母細胞のミトコンドリアDNAを減少させるための二分法の使用

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) ...