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I.ショウジョウバエの胚のコレクション<ol><li>卵のコレクションプレートを準備<ol><li> 8グラムの寒天を加えて200mlのdH 2 Oを沸かす</li><li> 50℃まで冷却</li><li> 2ミリリットルエタノール、2mlの酢酸を追加</li><li>トップス、35mmプラスチックシャーレに注ぐ</li><li>クールと4で気密容器° Cで保管</li><li>約80枚になります</li></ol></li><li>酵母ペーストを準備する<ol><li>貼り形成するために水にFleishmansアクティブベーキング酵母を溶かす</li><li>酵母を殺すために20〜30秒のためのマイクロ波（以上沸騰に注意） </li><li> 4℃で2週間で10 ccシリンジ（針なし）で保管して</li></ol></li><li>ハエ：卵の収集に使用される大人は、彼らは新鮮な食料へのアクセスがあったことを提供、3〜14日、新興の後に、一般的に最も生産的です。多くの変異株は、産卵とは、最も生産的となる年齢の変化の低下率で現れることができる肥沃度が低下している。一般的に、数百の大人は、良い産卵個体群ために作る。 </li><li>手順：卵のコレクション板に貼り付け酵母の薄層を広げて。これは、卵の産卵のための良好な基質を提供します。転送成人は、瓶の口の中にきれいな卵のコレクションのボトルとテープコレクションプレートに飛ぶ。湿度が上昇するとハエがペーストにし、瓶の側面にはまり込むように長く3-4時間よりも、これらのボトルにハエを放置しないでください。 </li></ol> II。ブリーチと胚Dechorionation <ol><li>アスピレーターに接続されているフィルターフラスコに接続したブフナー漏斗を設定します。漏斗でワットマン＃1枚の紙を置く。吸引を実行している状態で、滅菌蒸留水で紙を濡らし。 </li><li>洗浄瓶からの水の流れとろ紙の上にコレクションのプレートから胚を洗浄します。 </li><li>滅菌水のいくつかのボリュームでそれらをすすぎ、吸引器の電源をオフにし、50％の漂白剤の漏斗のボリュームを追加します。卵は5〜10分放置します。 chorionsは1〜2分で溶解されますが、長くて、漂白剤で胚を残す汚染酵母の多くは破棄されます。料理を洗い流したているすべての大人や幼虫を取り出す。 </li><li>滅菌蒸留水で滅菌ペトリ皿（NOT組織培養）に胚を洗浄します。それは事前に接液されていない場合の胚の多くは、皿の底に残ることでしょう。この料理の滅菌水で数回すすいでください。 </li><li>中期原腸胚期の胚を取り出すために透過光による胚を調べます。 </li></ol> III。単一の胚培養の準備<ol><li> DDM1を作る（セクションIVのレシピを参照してください） </li><li>直前の培養に胚の皿から水を注ぐ。滅菌メディアで胚をカバー。 </li><li> 3、35ミリメートルペトリ皿を設定します。各皿の4オートクレーブ円形カバーグラスに入れて。各カバースリップ上で、培地の5μlを置く。 Bellcoカバーガラス - 低鉛ガラスcoverlipsを使用してください。 Bellco生物学的ガラス製品 800-257-7043 猫＃1943から00012 </li><li>いくつかの非常に微細なピペットを引き出します。我々が使用するピペットはナリシゲPP - 83プラーに引っ張られている。ピペットの正確な寸法は重要ではなく、我々は通常、私たちが望むよりも細かそれらを引き出し、それらは大きさを測定するために胚のような視野の同じ分野で先端を折る。あなたが一度に胚のすべてを吸うしたくない、と自分は、それが細胞を吸うために5分かかるという情報が非常に小さくしたくない。両者の間のどこかを目指しています。透過光で胚を調べて、胚の内容を削除するには、ピペットに接続されている口の吸引管を使用してください。可能な限りカバースリップの近くに優しく細胞を追い出すことで、準備カバースリップ上に細胞を分散させる。あなたは、より高い電力でカバースリップを調査し、さらに同様の方法で細胞を分散させる場合があります。 </li><li>細胞は10分以上、もはや放置。洪水は、メディアと皿とインキュベーターに入れ、4-5％のCO 2環境、定義された培地を使用する場合。 22〜24℃の温度我々は日常的に23℃、湿度95％を使用。湿度は、蒸発を避けるためにしたいとして、重要です。あなたは23に設定された温度制御℃で冷凍庫に入れ、標準の哺乳類の培養器を使用することができます</li></ol> IV。文化を成長させるためのDDM1定義培地<ol><li> DMEMを加える：ハムのF12/DMEメディアの100 MLS（DMEM）（アーバインサイエンティフィック＃9052）に。 <ol><li> 0.476グラムのHepes（20mMの）（シグマ＃H - 3375）を追加します。ミックス。 </li><li> 1.25ミリリットル、L -グルタミン200mMのを（アーバインサイエンティフィック＃9317）を追加します。ミックス。 </li><li> 0.2μmの酢酸シリンジフィルター（2種類のフィルターを必要とする）とフードでフィルタリング。 </li><li> pHは6.64とOSM 288です。 </li><li> 15ミリリットルの遠心チューブに10ミリリットルのアリコートを行います。 </li><li> 4℃で2週間以上ないため。 注：常にメディアやサプリメントだけのために予約されているコンテナでオートクレーブ滅菌ろ過水を使用して行う。以下に記載されている追加補足：pH電極を置くか、またはMedia.To内の他の目的に使用される攪拌棒を絶対にDDM1を作るDMEM直前に培養することに。 </li></ol></li><li> DMEM追加の10〜MLS： <ul><li> 100μlのトランスフェリン</li><li> 100μlのプトレシン</li><li> 100μlのセレン</li><li> 100μlのプロゲステロン</li><li> 50μlのインスリン</li></ul></li></ol>

<ol>
	<li>Rohrbough, J., O'Dowd, D. K., Baines, R. A., Broadie, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+bases+of+behavioral+plasticity:+Establishing+and+modifying+synaptic+circuits+in+the+Drosophila+Genetic+System.">Cellular bases of behavioral plasticity: Establishing and modifying synaptic circuits in the Drosophila Genetic System.</a> J. Neurobiol. 54, 254-271 (2003).</li><li>O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-gated+currents+and+firing+properties+of+embryonic+Drosophila+neurons+grown+in+a+chemically+defined+medium.">Voltage-gated currents and firing properties of embryonic Drosophila neurons grown in a chemically defined medium.</a> J. Neurobiol. 27, 113-126 (1995).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+excitatory+synaptic+transmission+mediated+by+nAChR+in+cultured+Drosophila+neurons.">Fast excitatory synaptic transmission mediated by nAChR in cultured Drosophila neurons.</a> J. Neurosci. 19, 5311-5321 (1999).</li><li>Lee, D., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic-AMP-dependent+plasticity+at+excitatory+cholinergic+synapses+in+Drosophila+neurons:+Alterations+in+the+memory+mutant+dunce.">Cyclic-AMP-dependent plasticity at excitatory cholinergic synapses in Drosophila neurons: Alterations in the memory mutant dunce.</a> J. Neurosci. 20, 2104-2111 (2000).</li><li>Hodges, D. D., Lee, D., Boswell, K., Preston, C. F., Hall, L. M., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tipE+regulates+sodium-dependent+repetitive+firing+in+Drosophila+neurons.">tipE regulates sodium-dependent repetitive firing in Drosophila neurons.</a> Mol. Cell Neurosci. 19, 402-416 (2002).</li><li>Lee, D., Su, H., Dowd, O., K, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+receptors+containing+Rdl+subunits+mediate+fast+inhibitory+synaptic+transmission+in+Drosophila+neurons.">GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons.</a> J. Neurosci. 23, 4625-4634 (2003).</li></ol>

midgastrula期胚から調製ショウジョウバエの培養では神経細胞はin vitroで分化に先立って分割しその多くが神経芽細胞の前駆体、から生じる。このシステムは、このように神経発達のごく初期の段階（Rohrbaughら、2003）で重要な遺伝的要因と環境要因の探査のためのユニークな機会を提供します。我々は、単純な定義の培地中で増殖させた神経細胞でも（;リーとOダウド、1999 Oダウド、1995）機能的なシナプス結?...

このメディアに作られた文化は、約22〜24℃、5％CO2インキュベーター内で維持されなければならない我々は、インキュベーターが涼しい部屋に置き、標準の哺乳類の組織培養を使用し、23℃に加熱常に使用されるろ過水をオートクレーブし、メディアやサプリメントだけのために予約されている容器にしてください。 pH計を置いていないか、メディア内の他の目的に使用されるバーをかき混ぜることはありません。

preparation of neuronal cultures from midgastrula stage drosophila embryos

このビデオはmidgastrulaステージショウジョウバエの胚からの主ニューロン培養を行うための手順を示しています。漂白剤を使用して、胚とそのdechorionationを収集するための方法が実証されています。口の吸引管に接続されているガラスピペットを用いて、我々は、単一の胚からのすべてのセルの除去を示しています。コー​​ティングされていないガラスのカバースリップ上で培地の小さな（5リットル）ドロップに各embyroから細胞を分散させるための方法が示されている。めっき後の1時間での顕微鏡を使用してビューには、好ましい細胞密度を示しています。定義された培地中で増殖させた場合、生存細胞のほとんどは12-24時間で神経突起のプロセスを拡張する前に、文化に1回以上の分割神経芽です。顕微鏡を使用してビューには、神経突起伸長のレベルを示し、in vitroで2日間で健全な文化の中で期待分岐を。培養は22〜24℃、5％CO 2インキュベーター中で、定義された培地をベースにシンプルな炭酸で栽培されています神経突起のプロセスは、文化としたとき、彼らはしばしば機能的なシナプス結合を形成する隣接セルからの神経突起と接触するように最初の週にわたって詳しく説明し続ける。これらの培養物における神経細胞は、電位依存性ナトリウム、カルシウム、カリウムチャネルを発現しており、電気的興奮性があります。この培養系は、ニューロンの分化、興奮性、およびシナプス形成/機能を調節する分子遺伝学的要因と環境要因を研究するために有用です。

Watch this Scientific Journal Video about Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos at JoVE.com

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

このビデオはmidgastrulaステージショウジョウバエの胚からの一次ニューロン培養の準備を示しています。ライブ文化の景色は、重炭酸塩ベースの定義培地での生育の2日後に細胞を1めっき後の時間と分化した神経細胞を示す。神経細胞は電気的興奮性であり、シナプス結合を形成する。

Midgastrulaステージショウジョウバエの胚からの神経培養の準備

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

preparation-neuronal-cultures-from-midgastrula-stage-drosophila

Research

JoVE Journal

Biology

8.7K ビュー.  University of California, Irvine (UCI). このビデオはmidgastrulaステージショウジョウバエの胚からの一次ニューロン培養の準備を示しています。ライブ文化の景色は、重炭酸塩ベースの定義培地での生育の2日後に細胞を1めっき後の時間と分化した神経細胞を示す。神経細胞は電気的興奮性であり、シナプス結合を形成する。

ビデオ: Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

後期ステージショウジョウバエ蛹の脳からの初代神経培養

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

生物学

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

アーリーステージのニワトリ胚に成長因子コートビーズの配置

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HHステージ10ニワトリ胚のOVOエレクトロポレーションで

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

成長円錐の高分解能イメージングのためのアメフラシから神経細胞の培養

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

の調製ショウジョウバエ</emハンギングドロッププロトコルを使用してライブイメージングのための>胚

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

の準備アメフラシ感覚運動神経細胞の培養

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

ショウジョウバエの胚の生体内の遠心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

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 ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...