JBは、デイモンラニヨンがん研究財団フェローシップDRG - 1716 - 02サポートされていました。 JZは、NIH / NIGMSフェローシップF32 GM082174 - 02によってサポートされていました。 NPは、ハワードヒューズ医学研究所の研究者でもある。

1。フライケージの準備<ol><li>例えば32オンスの昆虫のカップとして便利な容器にショウジョウバエを （そのようなオレゴン- Rまたは遺伝子型などの野生型株）成長する。 </li><li>大胚コレクションケージに新しくeclosedハエを転送したり、人口のケージを飛ぶ。 </li><li> 〜25℃、湿度〜光12時間と暗12時間のサイクルで65％の同期胚の収集を容易にするための温度と部屋にケージを移動します。 </li><li>それらは糖蜜のプレートにストリーク酵母ペーストを殺した供給することによりケージ内のハエを維持する。 2-3日に一度糖蜜プレートを（殺した酵母ペーストで）変更するD. </li></ol> 2。同期胚のコレクション<ol><li>主細胞の準備の日に、1〜2時間プレレイアウト期間貼り殺さ酵母でコーティングWTH新鮮なプレートを飛ぶ餌。 </li><li>ちょうど12時間の明暗サイクルの前に、酵母ペーストの最小限の縞新鮮な糖蜜のプレートにあらかじめレイアウトプレートを交換してください。非同期古い胚を含むプレ素人のコレクションを、捨てる。 </li><li> 1〜2時間の期間のための胚を収集する</li><li> 5〜6時間にわたって25℃で胚や店舗の比較的同期人口を含むプレート° Cを削除します。胚は、この時点で高度な原腸胚の段階でとなります。 </li></ol> 3。胚の回収と洗浄<ol><li>胚は、下部の最高級のメッシュのふるいで収集するように、ウェットブラシでプレートから胚を緩め、そしてふるいの積み上げセットを介してなまぬるい水道水ですすぐ。できるだけ多くの酵母とハエの残骸として削除するには、数分間洗い流してください。 </li><li>ヒントは、胚が一面に蓄積するようにふるい、ゆっくりとdechorionationバスケットにふるいか​​らそれらを洗い流してください。 </li><li>胚を乾燥させることなく、組織培養のフードエリアに移動します。 </li></ol> 4。胚の均質化と細胞のプレーティング<ol><li> 5〜10分間50％（vol / vol）の漂白剤でそれらを浸漬することにより胚を滅菌しdechorionate。 70％（vol / vol）のエタノールでバスケットの壁から胚を洗浄します。また、エタノール処理の前に蒸留水で胚から漂白剤を洗浄する。この時点から以降の胚を滅菌条件下で組織培養フードで処理する必要があります。 </li><li>オートクレーブした蒸留水で漂白剤及び/またはエタノールを除去するために徹底的に胚を洗浄します。 </li><li> dechorionationステップの間、胚の量に基づいて、室温のM3媒体の適切な音量でダウンスホモジナイザーを埋める。胚の体積の間に、メディアのその比は0.5 mlの胚を超えることはできません：40 mlの培地（または〜100から200まで胚/ ml）を。 </li><li>水没胚のに十分なM3メディアの量を滅菌ビーカーでリンス胚を置きます。胚は2〜5分間浸してみましょう。 </li><li> dechorionationバスケットを分解して滅菌ペーパータオルでNitexメッシュが乾燥汚点。 </li><li>滅菌絵筆でホモジナイザーに胚を移す。 </li><li>すべての胚が中断されるまで、チューブの底に達することなく緩い乳棒を使用して、短いストロークで軽くホモジナイズ。ゆっくり、しかししっかりと、8個のフルストロークでホモジナイズする。それが上下に移動と乳棒をねじってはいけません。 </li><li>注ぐまたはピペッティングにより、滅菌50mlのコニカル遠心チューブにホモジネートを転送し、チューブをキャップ。 </li><li> 40グラム、ペレットに室温組織の破片、大規模な細胞塊と卵黄の膜で10分間遠心する。きれいなチューブに細胞と卵黄を含有する上清を移し、5分間の遠心分離のステップを繰り返します。 </li><li>慎重にペレットに、360グラムで10分間清浄なチューブやスピンに注ぐまたはピペッティングにより細胞を室温に上清を移す。上清を捨てる。培養液10ml中の細胞ペレット（RNAi実験のための無血清培地）を再懸濁します。 </li><li>細胞密度を推定する血球計数器を用いて生細胞を数える。一方、細胞をペレットに4.10を繰り返します。 </li><li>所望の濃度に細胞を再懸濁します。起動するには、1.7〜2.5 × 10 5細胞/ cm 2で1の範囲〜5 × 10 6細胞/ mlとプレートを試してみてください。 </li></ol> 5。 384ウェルプレートで培養した細胞のための主要な細胞のRNAi <ol><li> 384ウェルプレートにウェルあたり〜250 ngの最終濃度で各ウェルにdsRNAの5μlを分注する。 </li><li>マルチチャンネルピペットを使用して、各ウェルに異なるdsRNAを含む384ウェルプレートでの無血清M3培地中でウェル当たりの一次細胞を10ml（1〜4x106細胞/ ml）を分注する。また、細胞は、共焦点イメージングのための8ウェルガラスチャンバースライド上で培養することができます。 </li><li> 300グラム、室温で1分間プレートを遠心する。 25無血清培地での培養は、細胞℃で22時間または18でC ° 1-2のC D. </li><li>各ウェルに血清を含む培養液30mlを追加します。 </LI&gt; <li> 300グラム、室温で1分間プレートを遠心する。 </li><li> 〜7日25を追加5の湿度の高い室と文化初代細胞℃でプレートを置きます</li><li>画像セルは、ライブまたは免疫蛍光染色した後。 </li></ol> 6。代表的な結果： 最適な条件で、培養中の細胞は丸い形態と健康になります。文化は小さな細胞の破片を持っており、初期のメッキ後の50〜80％コンフルエントになります。これらの細胞は、長期間培養後形態学的変化を受けることになる。それは通常、完全に25℃に分化する培養中の細胞のために約5-8日かかる℃に良質初代培養は、通常、細胞 - の - の関心が文化の中で区別されているかどうかによって判断される。例えば、一次筋肉細胞は、多くの場合、枝のような形態を持っているし、サルコメアのシリーズで構成される筋原線維状のストライプと多核筋管となる。完全に分化した筋管は、通常、自発的に文化に移動します。対照的に、差別化された一次ニューロンは、それらの細胞体を持つクラスタを形成し、その軸索のケーブルと他のクラスタと接続する。 <img src="/files/ftp_upload/2215/2215fig1.jpg" alt="図1" /> 図1。初代細胞は、培養に播種された後、形態学的変化を受ける （A）384ウェルプレートのウェルに播種された後、右の初代培養細胞の明視野像。細胞の大部分はラウンドと無傷とうまく区切られている。 （B）初代培養細胞の明視野像めっき後の20時間。初代細胞は、すでに枝のような形態（長い矢印）を持つクラスタ（大きな矢印）と筋肉、この段階で認識することができますを形成する。 （C）5日間めっき後の初代培養の位相コントラスト画像を。文化は、神経細胞の拡張子（小矢印）、神経細胞のクラスター（ビッグ矢頭）と筋肉（中型矢印）で、はるかに高度に見えます。 <img src="/files/ftp_upload/2215/2215fig2.jpg" alt="図2" /> 図2。 dsRNAは治療を受けた初代細胞の例としては、384ウェルプレートで培養 初代細胞はDmef2 - GAL4を運ぶ胚、D42 - GAL4、UAS -水戸- GFPの導入遺伝子、これでDmef2 - GAL4と筋肉と運動神経の水戸- GFPのD42 - Gal4の駆動式、それぞれから単離された。細胞は、lacZの（AC）または膨張（あれば）（DF）のどちらかをターゲットにdsRNAは処理した。主な筋肉と神経細胞の両方は、GFP（A、C、D、F）で見ることができます。白い矢印は神経細胞の拡張子を指している。アクチンのファロイジン染色は（矢頭）のlacZ dsRNAは（BとC）で処理した培養での枝のような筋肉の構造を明らかにするのですが、それdsRNAは（EとF）で処理した培養でのラウンドアップ筋肉の形態。マージされた画像（C、F）で、筋肉は（白矢印）黄色の染色、しかし、赤色の負のショーのニューロンとその拡張されています。 <img src="/files/ftp_upload/2215/2215fig3.jpg" alt="図3" /> 図3。ヒトビトロネクチンコートしたカバーガラスをスライド上で培養した一次筋肉の共焦点顕微鏡写真 主な筋肉はimmunofluorescently抗ショウジョウバエタイチン（KZ）（マージの赤）とし、アクチンのファロイジン（マージの緑）で染色した。トップパネルには、野生型コントロールの筋肉であり、底部のものは、SAL（CG31374）短縮筋節とRNAiの筋肉です。

<ol>
	<li>Ashburner, M., Roote, J., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory culture of Drosophila. , 588-599 (1999).</li><li>Bai, J., Hartwig, J. H., Perrimon, N. S. A. L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+WH2-domain-containing+protein,+promotes+sarcomeric+actin+filament+elongation+from+pointed+ends+during+Drosophila+muscle+growth.">a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth.</a> Dev. Cell. 13, 828-842 (2007).</li><li>Bai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+Drosophila+primary+cells+for+genes+involved+in+muscle+assembly+and+maintenance+.">RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance .</a> Development. 135, 1439-1449 (2008).</li><li>Sepp, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+neural+outgrowth+genes+using+genome-wide+RNAi.">Identification of neural outgrowth genes using genome-wide RNAi.</a> PloS. Genet. 4, e1000111-e1000111 (2008).</li><li>Donady, J. J., Fyrberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+culturing+of+Drosophila+embryonic+cells+in+vitro.">Mass culturing of Drosophila embryonic cells in vitro.</a> TCA Manual. 3, 685-687 (1977).</li><li>Seecof, R. L., Alleaume, N., Teplitz, R. L., Gerson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+neurons+and+myocytes+in+cell+cultures+made+from+Drosophila+gastrulae.">Differentiation of neurons and myocytes in cell cultures made from Drosophila gastrulae.</a> Exp. Cell Res. 69, 161-173 (1971).</li><li>Bai, J., Sepp, K. J., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+Drosophila+primary+cells+dissociated+from+gastrula+embryos+and+their+use+in+RNAi+screening.">Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.</a> Nature Protocols. 4, 1502-1512 (2009).</li></ol>

利害の衝突は宣言されません。

ショウジョウバエの細胞の初代培養の発達パターンは、一次細胞調製の過程でいくつかの変数が原因の可能性が、大きく異なります。まず第一に、ハエ産卵のために使用される（古い週間以内）と健康（ウイルス性または細菌感染の自由）若いはずです。彼らは無用であり、それらの胚に由来する細胞が分化することができず、わずか1〜2日間、存続するとして、未受精卵または感染?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Shields and Sang M3 medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S3652</td>
<td>&nbsp;</td>
<tr>
<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>JRH Biosciences</td>
<td>12103-500M</td>
<td>&nbsp;</td>
<tr>
<td>Insulin from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I-6634</td>
<td>&nbsp;</td>
<tr>
<td>Large embryo collection cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-101</td>
<td>&nbsp;</td>
<tr>
<td>#25 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-454</td>
<td>&nbsp;</td>
<tr>
<td>#45 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-462</td>
<td>&nbsp;</td>
<tr>
<td>#120 US standard testing sieve</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>57334-474</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder, Wheaton 7 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>62400-620</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0040</td>
<td>&nbsp;</td>
<tr>
<td>Dounce tissue grinder,Kontes 40 mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>KT885300-0100</td>
<td>&nbsp;</td>
<tr>
<td>Costar, 384-well plate</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>29444-078</td>
<td>&nbsp;</td>
<tr>
<td>Lab-Tek II Chambered coverglass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>171080</td>
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primary cell cultures from drosophila gastrula embryos

ここでは、 ショウジョウバエの原腸胚からの解離の初代細胞を調製し、培養する方法を説明します。簡単に言えば、大量のは、若くて健康なハエから胚を上演、集め滅菌し、ガラスホモジナイザーを用いて、単一の細胞懸濁液にして物理的に解離されています。培地中の適切な密度で培養プレートまたはチャンバースライドに播種された後、これらの細胞は、さらにそれらの特定の細胞マーカーによって識別することができるいくつかの形態学的に異なる種類の細胞、に分化することができる。さらに、我々は、遺伝子ノックダウンを引き出すために二重鎖（ds）RNAをこれらの細胞を治療するための条件を提示する。 ショウジョウバエ初代細胞の効率的なRNAiは、単純にdsRNAを含有培養培地で細胞を浸すことによって達成される。他の分子、生化学、細胞イメージング解析とともに、効果的なRNAiの摂動を遂行する能力は、特にショウジョウバエ初代細胞、差別化された筋肉や神経細胞に関連するもので答えられるさまざまな質問ができるようになります。

Watch this Scientific Journal Video about Primary Cell Cultures from Drosophila Gastrula Embryos at JoVE.com

Primary Cell Cultures from Drosophila Gastrula Embryos

私たちは、から解離初代細胞を調製するための詳細なプロトコルを提供<em&gt; Drosophil</em&gt;胚。その他、分子生化学的および細胞イメージング手法と一緒に、効果的なRNAiの摂動を遂行する能力は、さまざまな質問がで対処できるようになります<em&gt;ショウジョウバエ</em&gt;一次電池。

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 ...

primary-cell-cultures-from-drosophila-gastrula-embryos

Research

JoVE Journal

Biology

Primary Cell Cultures from Drosophila Gastrula Embryos

13.8K ビュー.  Harvard Medical School. 私たちは、から解離初代細胞を調製するための詳細なプロトコルを提供 Drosophil胚。その他、分子生化学的および細胞イメージング手法と一緒に、効果的なRNAiの摂動を遂行する能力は、さまざまな質問がで対処できるようになりますショウジョウバエ一次電池。

ビデオ: Primary Cell Cultures from Drosophila Gastrula 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

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

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 ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

げっ歯類の新生児から一次分離した中脳ドーパミン細胞培養

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

げっ歯類からプライマリ大人の線維芽細胞培養の確立

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

キネシンの単離及び精製からショウジョウバエ胚

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

初代ヒト線維芽細胞培養からナイーブ成人幹細胞の分離

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...