この研究は、テキサスA＆M大学からスタートアップ資金の一部で賄われていた

解剖前の準備 手術器具：オートクレーブによるすべての解剖はさみや鉗子を滅菌 カバーのクリーニング： <ol><li>少なくとも24時間、33％塩酸で1L草のビーカー内のすべてのカバースリップを（24ウェルプレート用の直径15mm）をソーク</li><li>すべての残留HClを除去するために時折揺れで10分間流水で洗う</li><li>ミリポア水で洗い流してください</li><li>水を切る</li><li> 95から98パーセントのエタノールでカバーガラスを浸し</li><li>平板と空気のきれいなティッシュにカバースリップを転送するには、カバースリップを乾燥させる</li><li> 、ガラス製ビーカーにアルミ箔とオートクレーブでカバーをカバースリップを転送する 注：カバースリップをこの段階で保存することができます。 </li><li>培養プレートに、個々のカバースリップを配置。コー​​ティングのための準備</li></ol> カバースリップコーティング： <ol><li> PBSとポリ- D -リジン（-20アリコート° Cとして格納されているPBS、0.5％のPDL、10 mg / mlのウシ血清アルブミン）の100 ×ストックを希釈し、フィルター滅菌（0.22μm）で</li><li> 37℃2時間× 1：PDLソリューション（〜0.15ミリリットル/ cm 2で ）°インキュベーターでCとコートのカバースリップ</li><li> PDLソリューションを取り外して、完全無菌のddH 2 Oと、乾燥カバーグラスで3回洗浄</li></ol> 注：各節郭清のためのコート十分なカバーグラス/プレート。 PDLでコーティングされたカバースリップは4℃で数週間保存することができるメディアとソリューション 解剖の培地（DM）は：10mMのHEPES（インビトロジェン15630）を添加した滅菌、氷冷ハンクス平衡塩溶液（W / OのCa 2 +、Mg 2 +を ）（HBSS、インビトロジェン14175）を使用してください。 集計培地：2％B27、1 ×佐藤、0.5mMのピルビン酸ナトリウム、0.75mmのグルタミン、60μg/mlN -アセチルシステイン、5μg/ mlの：DMEM/NBB27培地はDMEM / Neurobasal（巻1時01巻）が含まれているインスリン、10nmのd -ビオチンおよび1％ペニシリン/ストレプトマイシン。凝集培養培地100mlを作るために、50 mlのDMEM（W / Oピルビン酸/グルタミン、インビトロジェン11960）、50 mlのNeurobasal培地（Invitrogen 211034）、375μlのグルタミン（100 ×、インビトロジェン35050）、5.5 mgのピルビン酸ナトリウムを混合（シグマP2256）、2 mlのB27（インビトロジェン17504）、6.3 mgのN -アセチルシステイン（シグマA8199）、佐藤株式を1 ml（100 ×ストック、下記参照）、d -ビオチン株式25μlの（4000 ×ストック、40 -20分注して保存されているPBSでμM℃のシグマB4639）、インスリン100μlの（1000 ×株式、-20分注し° C、シグマ16634として保存されている0.01N塩酸で5 mg / ml）を、1 mlのペニシリン/ストレプトマイシン（100 ×株式、インビトロジェン15140）は、ろ過滅菌し、4℃で保存します。 佐藤100 ×ストック溶液：株式の40mlを行うには：400mgのアポトランスフェリン（シグマT2252）、400 mgのBSA（シグマA9647）、として保存されているプロゲステロンの10μlの（25 mg / mlのエタノールとの混合40mlのNeurobasal -20分注し℃のシグマP8783）、64 mgのプトレシン（シグマP7505）、および亜セレン酸ナトリウム40μlの（-20アリコートとして保存されているPBSで30μM、° C、シグマS5261）。 -20℃でアリコートとして佐藤ストック溶液、および店舗を消毒フィルタリング ニューロンのメッキ培地（PM）：Neurobasal培地、2％B27、2mMグルタミン（100株、-20℃、Invitrogen社製25030に格納されているアリコート）、25μmのグルタミン酸（100株、-20℃で保存したアリコート）および1％ペニシリン/ストレプトマイシン。 ニューロン培養培地（CM）：Neurobasal培地、2％B27、2mMグルタミン（100株、-20℃で保存したアリコート）及び1％ペニシリン/ストレプトマイシン。 5 - FDU株式：Neurobasal培地で100倍の株式、1mMの5 -フルオロ-2&#39; -デオキシウリジン（Sigma社F0503）and1mMウリジン（Sigma社U3003）。フィルタは、-20℃で滅菌してアリコートとして保存 パパインの消化液（前の節郭清に新鮮なように）： <ol><li> 4ミリリットルDMで3.2 mgのL -システイン（シグマC - 7352）を溶かす</li><li> 37℃で1NのNaOH（pH試験ストリップのテスト）と水浴の場所で約7.4にpHを調整する° C 注意：（下記参照）、右組織消化する前に次のステップを実行します。 </li><li> 20単位/ mlの最終濃度にパパインを追加</li><li> （0.22ミリメートル）滅菌フィルター処理、および37℃の水浴中で場所° C </li></ol> トリプシン阻害剤溶液（前の節郭清に新鮮なように）： <ol><li> 20ミリリットルDMで0.2グラムトリプシンインヒビター（Sigma社T7295）を溶かす</li><li> pHをチェックし、1 N NaOHで〜7.4にpHを調整する</li><li>滅菌フィルター処理、および37℃の水浴中で場所° C </li></ol>脳の解剖 セットアップ： <ul><li>滅菌鉗子とはさみ</li><li> 70％エタノール</li><li>クリーンティッシュペーパーとdiapベンチで二パッド</li><li>ベンチで子宮や胎児のための氷冷DMと10cmのペトリ皿</li><li>解剖顕微鏡で氷のプラットフォーム</li><li>氷の上にDMを2〜3 6センチメートル料理</li><li> 37℃の水浴中で暖かいPM </li></ul><ol><li>あなたの動物実験によって承認された手順と連絡委員会（IACUC）によると妊娠したSprague - Dawleyラット（E16）を安楽死させる</li><li>腹部領域の上におむつパッドと去勢のアルコールのラットレイ</li><li>皮膚のみをカットするV字型の切開を行うために腹部の皮膚とはさみのペアを保持するためにピンセットを使用して、 </li><li>皮膚を広げますと筋肉層を介してカットして別の滅菌ピンセットやハサミを使う</li><li>胚嚢の連鎖を抽出し、氷冷DMを含む10cmの皿にそれらを転送する</li><li>マイクロダイセクションのはさみを使用して、慎重に氷のプラットフォーム上でDMときれいな10cmの皿に嚢と各胚から脳、そして場所解放脳から各胚を削除</li><li>層流フード内や解剖顕微鏡下で、中脳/後脳のセクションを削除し、髄膜を除去するために微細な鉗子を使用し、氷のパックにDMで新しい6 cmディッシュに洗浄皮質/海馬を転送 注：この段階で、組織は酵素消化の準備ができている。 </li><li>パパインとトリプシンの阻害剤溶液およびフィルター滅菌を行う</li><li>皮質/海馬からDM過剰を取り除く</li><li>準備パパイン消化溶液の4ミリリットルを追加します。 </li><li> 50ミリリットルチューブに、すべてのコンテンツを転送し、37℃の水浴中にファルコンチューブを配置° Cを正確に5分間</li><li>ピペットでパパイン溶液を除去</li><li> 2〜3分間5ミリリットルトリプシン阻害剤溶液、渦巻管、および37℃の水浴中で行わ° ​​Cを追加</li><li>トリプシンインヒビターの溶液を除去</li><li>ステップ13と14を3回繰り返します</li><li> 20ミリリットル暖かいPMを追加</li><li>すべての塊が消失するまでひいて粉にする（〜20〜30倍） </li><li> 7分のために100xgで遠心分離</li><li> 10ミリリットルPMでペレットを再懸濁し、遠心分離により2回洗浄する</li><li>定期的なニューロンの培養のためにまたは集約培養用のNBB27/DMEMで10ミリリットルPMでペレットを再懸濁します</li><li> 70μmセルふるいを介して細胞を渡す</li><li>生細胞を数える</li><li>定期的なニューロンの培養用細胞を以下に記載または板として集約文化に進みます 注：このステップでは、高濃縮ニューロンの培養は、実験の目的に応じて2 200〜640細胞/ mmの密度で、PDLコーティングプレート上に解離細胞を播種することによって達成することができます。細胞は（〜2 - 4H）接続した後は、暖かい日に培地を交換してください。長時間培養した場合、4日目に、暖かいCMで培地の半分を交換してください。非常に神経細胞の培養、精製するため、有糸分裂阻害剤5 - FDUは（最終濃度10μM）の非神経細胞の増殖を（すなわち、DIV2 - 3の間）を阻害するDIV2で追加することができます。 2日間のCMで回復した後、細胞は、パルスは完全培地の変化に続いて別の2日間（DIV 6-7）のために5 - FDUで再び処理されます。その後、3〜4日ごとに新鮮な、温かみのあるCMで培地の半分を交換してください。 4週間までのニューロンは、実行可能です。 </li></ol>凝集体の調製およびメッキ<ol><li> 2 × 10 6細胞/ mlの密度で1 ×佐藤、10ng/ml CNTFおよび10μMフォルスコリンを含むNBB27/DMEM培地で解離細胞をサスペンド</li><li>コー​​ティングされていない6ウェルプレートの各ウェルに細胞懸濁液の2ミリリットルを転送する</li><li> 3泊の培養。毎日、穏やかに一度P1000 Pipetmanを用いて細胞を再懸濁 注：セルの集合体（図1）を形成し始める。 </li><li>マトリゲルと集約メッキ、コートプレート/カバースリップの前日： <ul><li>株式マトリゲル（成長因子が減少し、BD Biosciences社354230）氷の上に冷たいDMEMで1:20に希釈する 注：マトリゲルアリコートを、製造業者のプロトコルごとに用意する必要があります。すべてのヒントを、エッペンドルフチュー​​ブとストックのアリコートを作るためのソリューションは、マトリゲル重合を避けるために、寒いはずです。マトリゲルのアリコートを-80℃に維持し、4℃で解凍されています</li><li> 37℃インキュベーターで一晩希釈マトリゲル溶液を300μl/wellでコートは、以前にPDLコートしたカバースリップ</li><li>温かいPBSで洗い、その後暖かい滅菌のddH 2 Oでプレートを洗浄し、インキュベーターでプレートを残す。 </li></ul></li><li>凝集体形成後の3日間、再び静かに細胞を再懸濁し、ふるい50ミリリットルファルコンチューブに200μmのメッシュを介してサスペンション。凝集体は重力（〜3 - 5分）でチューブの底に沈降することができます</li><li>注意深く上清を除去</li><li>培地を加え、穏やか凝集体を再懸濁し、それらを再び沈殿ができます。死んだ細胞、個々の細胞および残骸を削除する場合は、この手順を数回繰り返します。上清はまだ非凝集細胞とdebriが含まれているかどうかを確認するために顕微鏡を使用して、の</li><li>ゆっくりと同じNBB27/DMEM媒体（図1）、カウントの集計に集計を再懸濁します。 </li><li> 25から30の周りaggreggats/50ulに凝集体の密度を調整する</li><li>メッキのための2ミリリットルエッペンドルフチュー​​ブに集約サスペンションの転送アリコート（500 -1000μl） 注：集合体は、チューブの底に落ち着く傾向があるので、それはメッキのために集約サスペンションの複数のチューブを作ることが重要です。ゆっくりと各カバースリップ上に播種した凝集体の同じような数を確保するためにコーティングされたカバースリップまたは培養プレート上にそれらを播種する前に、しばしば凝集体を懸濁します。 </li><li>インキュベーターからマトリゲルでコーティングされたカバースリップを含む培養プレートを取り出してください。各ウェルから溶液を除去、温かいPBSとのddH 2 Oでカバーグラスを洗浄し、乾燥簡潔に。彼らは現在集計播種の準備ができている</li><li>それらは播種前に均等に分散されるように集約懸濁液を含むチューブを反転。各カバーの中央に集約し懸濁液50μlずつをロードし、ゆっくりと凝集体がカバースリップに均等にアタッチできるようにそれ以上の障害なくインキュベーターにバックプレートを置く 注：集合体の密度が非常に重要です。凝集体を播種している場合はあまりにも接近したり、播種密度が高い場合、彼らは成長するにつれて一緒にマージする傾向がある。 </li><li>翌朝以降早期に各ウェルに4-6時間に追加のDMEM/NBB27medium（500μl）を添加</li><li>集計文化を維持するために、ハーフ3〜4日ごとに培地を変更してください。培地を変更すると、凝集を妨害しないように注意してください、これは穏やかにウェルの側壁に沿ってメディアを追加することによって行われます。 注：数時間プレートに付着凝集後、凝集体からの神経突起の成長を観察することができる。軸索は、最初の2週間で急速に成長し、凝集体（図2）の間に豊富な接続を形成する。グリア前駆細胞は、凝集体から放射状に移動し、星状膠細胞と成熟オリゴデンドロサイトに時間をかけて分化する。 </li></ol><img src="/files/ftp_upload/1304/1304fig1.jpg" alt="図1" /> 図1洗浄後に別の日と集計で凝集体を形成する細胞の位相コントラスト像。 <img src="/files/ftp_upload/1304/1304fig2.jpg" alt="図2" /> 2〜12日間マトリゲルでコーティングされたカバースリップ上に播種した後の骨材文化の図2。位相コントラスト像。

<ol>
	<li>Matthieu, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+in+rat+brain+aggregating+cell+cultures.">Myelination in rat brain aggregating cell cultures.</a> Neuroscience. 3, 565-56 (1978).</li><li>Chen, Y. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+and+mouse+oligodendrocyte+precursor+cells.">Isolation and culture of rat and mouse oligodendrocyte precursor cells.</a> Nat. Protocols. 2, 1044-10 (2007).</li><li>Watkins, T. A., Emery, B., Mulinyawe, S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stages+of+myelination+regulated+by+gamma-secretase+and+astrocytes+in+a+rapidly+myelinating+CNS+coculture+system.">Distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating CNS coculture system.</a> Neuron. 60, 555-55 (2008).</li><li>Svenningsen, A. F., Shan, W. e. i. -. S. o. n. g., Colman, D. a. v. i. d. R., Pedraza, L. i. l. i. a. n. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+method+for+culturing+embryonic+neuron-glial+cell+cocultures.">Rapid method for culturing embryonic neuron-glial cell cocultures.</a> Journal of Neuroscience Research. 72, 565-56 (2003).</li></ol>

初期の研究は、シナプスと回転を介したフリーフローティング集約文化1の成熟したミエリンの形成を報告した。ここで説明CNSの集合体の培養系は、細胞の開発、マイグレーションおよびin vitroでの分化の解析を容易にするために、従来の2D文化の利便性を持つ三次元凝集体における多能性前駆細胞の無血清成長を兼ね備えています。システムは、神経前駆体のために修正...

preparation of rat brain aggregate cultures for neuron and glia development studies

中枢神経系（CNS）における成熟した神経細胞とグリア細胞への反復する前駆細胞の発生や分化は、軸索 - グリア相互作用、多能性前駆細胞の性質と分化、およびオリゴデンドログリアの進行を調査する神経科学者のための強力なプラットフォームを提供することをin vitroの系細胞および分子レベルでの系統細胞。ここでは3-4週間に無血清培地に維持することができるとニューロン - グリア相互作用と中枢神経系の髄鞘形成を研究するモデルとして我々の研究室で使用されているラット胚のforebrains、からCNSの集合体培養系を説明します。このビデオクリップは、E16ラット脳からこれらのCNSの集合体の文化を分離し、成長させる方法を紹介します。さらに、同じ脳の解剖から、高濃縮レギュラー解離ニューロン培養を容易に得ることができると、様々な研究に使用CNSニューロンや他の細胞との共培養に使用されます。

Watch this Scientific Journal Video about Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies at JoVE.com

Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

胎児ラット脳の集合体の培養系のためのプロトコルが記述されています。凝集体の多能性前駆細胞は、開発し、神経細胞、アストロサイト、オリゴデンドロサイトに分化することができる。

ニューロンとグリアの開発研究ラットの脳の集計文化の調製

An in vitro system that recapitulates the development and differentiation of progenitors into mature neurons and glia in the central nervous system (CNS) ...

preparation-rat-brain-aggregate-cultures-for-neuron-glia-development

Research

JoVE Journal

Biology

12.3K ビュー.  Texas A&M University (TAMU). 胎児ラット脳の集合体の培養系のためのプロトコルが記述されています。凝集体の多能性前駆細胞は、開発し、神経細胞、アストロサイト、オリゴデンドロサイトに分化することができる。

ビデオ: Preparation of Rat Brain Aggregate Cultures for Neuron and Glia Development Studies

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 ...

ALS - Motor Neuron Disease: Mechanism and Development of New Therapies

Jeffrey D. Rothstein speaks about the pathology and mechanisms underlying amyotrophic lateral sclerosis or ALS, advances in ALS research, and current strategies towards the development of therapies.

ALS - 運動ニューロン疾患：新しい治療法のメカニズムと開発

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

ニューロン相馬と軸索のコンパートメントのためのマイクロ流体デバイスの作製

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Preparation of Dissociated Mouse Cortical Neuron Cultures

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures can be used for a variety of applications including immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging, protein and/or RNA isolation. These cultures also provide a platform to study the neuronal development of transgenic animals that carry a late embryonic or postnatal lethal gene mutation. The procedure is relatively straight forward, requires some experience in tissue culture technique and should not take longer than two to three hours if you are properly prepared. Careful separation of the cortical rind from the thalamo-cortical fiber tract will reduce the number of unwanted non-neuronal cells. To increase yields of neuronal cells triturate the pieces of the cortical tissue gently after the enzyme incubation step. This is imperative as it prevents unnecessary injury to cells and premature neuronal cell death. Since these cultures are maintained in the absence of glia feeder cells, they also offer an added advantage of growing cultures enriched in neurons.

This video shows a procedure for generating neuronal cultures from late embryo and early postnatal mouse cortex. These cultures can be used for immunocytochemistry, biochemistry, electrophysiology, calcium and sodium imaging and provide a platform to study the neuronal development of transgenic animals that carry a postnatal lethal gene mutation.

解離マウス皮質ニューロンの培養の準備

This video will guide you through the process for generating cortical neuronal cultures from late embryo and early postnatal mouse brain. These cultures ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

This video demonstrates the dissection and removal of the fetal thymus as well the preparation of ex vivo cultures of 2-dGuo-treated thymus.

2 - dGuo処理した胸腺器官培養の準備

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

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 ...

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

マルチコンパートメントのCNSニューロングリア共培養マイクロ流体プラットフォーム

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

から毒素誘導とタンパク質抽出フザリウム属属。</emプロテオーム研究>文化

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Preparation of Rat Tail Tendons for Biomechanical and Mechanobiological Studies

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation.
Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies. Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.
At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced. Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor.
Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons.

This article describes the experimental procedures used to prepare rat tail tendons for biomechanical and mechanobiological studies. Several features of the main steps in preparation are demonstrated, beginning with extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber.

生体力学とMechanobiological研究ラット尾腱の準備

Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working ...