我々は、プロトコル上で貴重なコメントを提供するためポッターのグループのメンバーに感謝したいと思います。 、この作品は、神経財団のアメリカアカデミーからCMHへの臨床研究研修フェローシップ、一般医科学研究所（NIGMSからフェローシップによって賄われていた<a href="http://www.nigms.nih.gov/">http://www.nigms.nih.gov/</a> JDRへ（GM08169） ）、神経疾患のNIH国立研究所からの助成金とストローク（NS054809）、国際緊急援助隊（NS060392）、NSF EFRI助成金（0836017）、コールター財団トランスレーショナルリサーチ賞、とのルースL.キルシュシュタイン国立リサーチサービス賞JDRのトランスレーショナルリサーチのフェローシップ（NS007480）。

<ol>
	<li>Potter, S. M., DeMarse, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+neural+cell+culture+for+long-term+studies.">A new approach to neural cell culture for long-term studies.</a> J Neurosci Methods. 110, 1-2 (2001).</li><li>Rambani, K., Vukasinovic, J., Glezer, A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+thick+brain+slices:+an+interstitial+3D+microperfusion+system+for+enhanced+viability.">Culturing thick brain slices: an interstitial 3D microperfusion system for enhanced viability.</a> J Neurosci Methods. 180, 243-254 (2009).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. 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J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+and+growth+of+hippocampal+neurons+in+defined+medium+at+low+density:+advantages+of+a+sandwich+culture+technique+or+low+oxygen.">Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen.</a> Brain Res. 494, 65-74 (1989).</li><li>Lelong, I. H., Petegnief, V., Rebel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cells+mature+faster+on+polyethyleneimine+coated+plates+than+on+polylysine+coated+plates.">Neuronal cells mature faster on polyethyleneimine coated plates than on polylysine coated plates.</a> J Neurosci Res. 32, 562-568 (1992).</li><li>Potter, S. M., Pine, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+transplant+staining+with+DiI+and+vital+imaging+by+2-photon+laser-scanning+microscopy.">Neural transplant staining with DiI and vital imaging by 2-photon laser-scanning microscopy.</a> Scanning Microsc Suppl. 10, 189-199 (1996).</li><li>Rolston, J. D., Gross, R. E., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low-cost+multielectrode+system+for+data+acquisition+enabling+real-time+closed-loop+processing+with+rapid+recovery+from+stimulation+artifacts.">A low-cost multielectrode system for data acquisition enabling real-time closed-loop processing with rapid recovery from stimulation artifacts.</a> Front Neuroengineering. 2, (2009).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+all-channel+stimulator+for+electrode+arrays,+with+real-time+control.">A versatile all-channel stimulator for electrode arrays, with real-time control.</a> J Neural Eng. 1, 39-45 (2004).</li><li>van Pelt, J., Wolters, P. S., Corner, M. A., Rutten, W. L., Ramakers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+characterization+of+firing+dynamics+of+spontaneous+bursts+in+cultured+neural+networks.">Long-term characterization of firing dynamics of spontaneous bursts in cultured neural networks.</a> IEEE Trans Biomed Eng. 51, 2051-2062 (2004).</li><li>Wagenaar, D. A., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extremely+rich+repertoire+of+bursting+patterns+during+the+development+of+cortical+cultures.">An extremely rich repertoire of bursting patterns during the development of cortical cultures.</a> BMC Neurosci. 7, (2006).</li><li>Wagenaar, D. A., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+multi-channel+stimulus+artifact+suppression+by+local+curve+fitting.">Real-time multi-channel stimulus artifact suppression by local curve fitting.</a> J Neurosci Methods. 120, 113-120 (2002).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+activity-dependent+plasticity+of+action+potential+propagation+delay+and+amplitude+in+cortical+networks.">Long-term activity-dependent plasticity of action potential propagation delay and amplitude in cortical networks.</a> PLoS One. 3 (5), e2088-e2088 (2008).</li><li>Wagenaar, D. A., Madhavan, R., Pine, J., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+bursting+in+cortical+cultures+with+closed-loop+multi-electrode+stimulation.">Controlling bursting in cortical cultures with closed-loop multi-electrode stimulation.</a> J Neurosci. 25 (3), 680-688 (2005).</li><li>Jimbo, Y., Tateno, T., Robinson, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+induction+of+pathway-specific+potentiation+and+depression+in+networks+of+cortical+neurons.">Simultaneous induction of pathway-specific potentiation and depression in networks of cortical neurons.</a> Biophys J. 76 (2), 670-678 (1999).</li><li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing nerve cells. , (1998).</li></ol>

CMHは、開示には関係ありません。<br /&gt; JDRとSMPは、アクシオンアプライドバイオシステムズのコンサルタントが支払われます。<br /この記事とビデオ用&gt;投稿料およびビデオ処理手数料は、丁重にマルチチャンネルのシステムによって後援されています。

このプロトコルは、どのように文化と同様に記録し、神経ネットワークを刺激への導入をマイクロ電極アレイ上の神経ネットワークを維持する上での指示を示しています。多国間環境協定から録音するより一般的な問題の一部は、MEAのトラブルシューティングのセクションで議論された。で説明すると、しばしばこれらの変更が特定の実験設計で必要とされる特定の条件を最適化するために利用されるプロトコルで、時々のバリエーションがあります。 ここで紹介する記録と刺激的なシステムがMCSのプリアンプでNeuroRighter 15を設計したカスタムで構成されていますが、神経回路網とのインタフェースを提供できる他の複数のシステムがあります。特定のセットアップを選択すると、特定の実験のニーズに依存します。 単純な記録と刺激の例は、しかし、MEAの上にこれらの培養物は、シナプス活性と神経回路網の接続に関する複雑な問題への洞察を提供するために使用できるこのプロトコルで提供されていました。上記のように、進行中の仕事は、細胞可塑性、開発、興奮毒性および神経変性のようなプロセスを研究するためにこの技術を利用している述べた。6-10は例えば、我々の研究室から最近の出版物はでリアルタイム閉ループの刺激とin vitroでの学習の指示再現可能な目標を示した現在の神経回路網の活動3に応答。 MEAの文化はしばしば凝り性で繊細なものですが、そのシンプルさとアクセシビリティ（インタクトな動物と比較して）のおかげで、彼らはネットワークレベルでの神経活動を研究するための強力なモデルを提供しています。

電源<ul><li>使い捨てボトルトップフィルター（150ミリリットル、0.2μmの細孔径; Nalgene＃565〜0020） </li><li>胎生18日時限妊娠雌ラット</li><li>微小電極アレイ（B1、プロトコルの説明を参照） </li><li>微小電極アレイの蓋（プロトコル、B3の説明を参照してください） </li><li>ナイロンセルストレーナー（40μmの、BDファルコン、＃352340） </li><li>ポリプロピレンコニカルバイアル（無菌、15ミリリットル） </li><li>ポリスチレンシャーレ（滅菌、35mmと100x15mm） </li><li>ピペットチップ（滅菌、20μlの、200μlのと1000μl） </li><li>血清学的ピペット（5ml）を</li></ul>設備： <ul><li>生物学的安全キャビネット/層流フード</li><li>セルカウンター</li><li>遠心</li><li>氷の容器</li><li>解剖ハサミや鉗子（滅菌） </li><li>層流フードの解剖顕微鏡</li><li>ハンドヘルド電気pipetman </li><li>血球計</li><li>イソフルランとガス室</li><li>光顕微鏡</li><li>ハンドヘルドpipetman（P - 20（20μlの）、P - 200（200μlの）、P - 1000（1000μl）） </li><li> MEA記録方式（プロトコルの説明を参照して、D1） </li><li>げっ歯類のギロチン</li><li>トライガスインキュベーター（プロトコル、B15の説明を参照してください） </li><li>フードに取り付けられアスピレーターで真空フラスコ</li><li>渦</li><li>水浴（35〜37 ° C） </li></ul>ソリューションと試薬： BSA溶液： 5％を作るリン酸のBSA（シグマ9418）は、生理食塩水pH7.4の緩衝化し、0.2μmのでろ過。このソリューションは、事前に準備され、数ヶ月間、4℃で保存することができる。 セルメディア： 90ミリリットルダルベッコ改変イーグル培地（アーバインサイエンティフィック9024）、10mlのウマ血清（ギブコ16050）、1mlのピルビン酸ナトリウム（100mMの、ギブコ11360）、250μlのグルタミン（ギブコ35050）、及びインスリンを（最終濃度2.5μg/ mlの、組み合わせるシグマI - 5500）は、0.2μmのfilter.22ウォームを通過し、使用前にインキュベーターで細胞の培地を平衡化してください。 パパイン溶液を調製するための解剖ソリューション：23 <table border="1"><tr><td>解剖液（100ミリリットル株） </td><td> 4で、順番に0.22μmのフィルターと店舗を組み合わせた℃に</td><td></td></tr><tr><td>コンポーネント</td><td>ボリュームミリリットル</td><td>最終濃度（mM）を</td></tr><tr><td>ダブル蒸留水</td><td> 91.175 </td><td></td></tr><tr><td> 1 M MgCl 2の </td><td> 0.58 </td><td> 5.8 mMの</td></tr><tr><td> 1 M CaCl 2を </td><td> 0.025 </td><td> 0.25 mMの</td></tr><tr><td> 0.5 MのHEPES </td><td> 0.32 </td><td> 1.6mmの</td></tr><tr><td> 0.5パーセントフェノールレッド</td><td> 0.2 </td><td> （0.001％） </td></tr><tr><td> 0.1N NaOHを</td><td> 0.2 </td><td> 0.2mMの</td></tr><tr><td> 2 M硫酸ナトリウム</td><td> 4.5 </td><td> 90 mmの</td></tr><tr><td> 1 M K2SO4 </td><td> 3.0 </td><td> 30mMの</td></tr><tr><td> 0.1 Mキヌレン酸</td><td> 1.0 </td><td> 1mMの</td></tr><tr><td> 5mMのAPV </td><td> 1.0 </td><td> 0.05mMの</td></tr></table>このソリューションは、事前に準備され、数ヶ月間、4℃で保存することができる。 DNアーゼI： インビトロジェン（＃18047019）。 50μlのアリコートを-80℃で凍結点滅し、保存されていますアリコートを細胞解離のプロセスを開始する前に、層流フードで解凍されています。 ハンクス平衡塩溶液： シグマ（＃4891）。 1X溶液を滅菌0.2μmのでフィルタリングし、4℃で保存したが、脱イオン水中で調製され Hibernateのソリューション： 培養の日に準備する。 2ミリリットルB - 27サプリメント（50X、Invitrogen社＃17504）、0.2μmのフィルターで98ミリリットル、L - 15培地（インビトロジェン11415）を組み合わせる。 ラミニンのソリューション： 培養の日に準備する。 0.02mg/mlの最終濃度980μLの細胞培地中でラミニンの20μlのを（1mg/ml、Sigma社L - 2020）で希釈します。ラミニン（1mg/ml）を20μlのアリコートを-80℃で保存し、使用直前に解凍されています。急速解凍は、時々ラミニンのゲル化を招く可能性が融解はベンチの上ではなく、水浴中で発生する必要があります。 パパイン液：23 解剖のソリューションの暖かい10ミリリットル（上記）30〜32℃まで200台パパイン（シグマP - 4762）および1.6 mgのL -システイン（シグマC - 7880）を追加します。 0.1 N NaOHを約14μlを7.4にpHを調整する。ソリューションのCLまで約30分間インキュベート耳、0.2μmのフィルター、-80℃で15 mlのポリプロピレン製滅菌コニカルバイアルとストアの2 mlのアリコートにおける液体窒素でフラッシュ凍結℃の° Cより前のプロトコルを開始するまで35から37にアリコートを解凍。 ポリエチレンイミン（PEI）解決策：13 PEIの100μlの（50％w / vの、シグマP - 3143）が0.05％の最終濃度w / vのために100mlのホウ酸ナトリウム緩衝液（0.1 M pHは8.4、シグマB - 9876）で希釈し、 0.2μmので無菌フィルター。これは、事前に準備され、月間4℃で保存することができる。 滅菌脱イオン水： オートクレーブし、使用前に室温まで冷却することができます。 トリプシン（EDTA、ギブコ25200 0.25％）： 1ミリリットルのアリコートを-80℃で凍結点滅し、保存されていますアリコートは、使用前に35から37℃の水浴中で解凍されています。

how to culture, record and stimulate neuronal networks on micro-electrode arrays (meas)

最後の世紀のために、世界中の多くの神経科学者は神経回路網がどのように働くかを理解するために命を捧げていると、なぜ彼らは様々な疾患に動作しなくなるものがあります。研究は、神経病理学的観察、遺伝標識と蛍光顕微鏡、および解離神経細胞やスライス標本の両方で細胞内記録を含めました。このプロトコルは、マイクロ電極アレイ（マルチ電極アレイも呼ばれ、MEAの）上に成長解離ニューロン培養を伴う別の技術を、説明します。 他の技術よりもこのシステムを使用する複数の利点があります。多国間環境協定上の解離ニューロン培養では、ネットワークアクティビティがアレイの複数の電極を介して電気刺激シーケンスで操作可能な単純化したモデルを提供しています。ネットワークが小規模であるため、刺激の影響はそのまま調剤の場合ではない、観測可能な領域に制限されています。細胞は様々なイメージング技術を使用して監視する形態の変化が容易に単層に成長する。最後に、多国間環境協定上の文化は他の培養技術に固有の任意の明確な時間の制限を削除するin vitroで一年以上生き延びることができる1。 世界中の私たちの研究室や他の人は神経回路網に関する重要な質問をするこの技術を利用しています。このプロトコルの目的は、世話、セットアップから録音すると電気的に多国間での文化を刺激するために必要な情報を提供することです。ネットワークで生理学的に関連する質問をするとのより良い理解につながる細胞レベルのための手段を提供するin vitroでのネットワークでは脳機能と機能不全。

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How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

このプロトコルは、セットアップの世話から、記録と電気的に多国間での文化を刺激するために必要な情報を提供します。<em&gt;試験管内で</em&gt;ネットワークは、脳機能と機能障害のより良い理解につながるネットワークと細胞レベルでの生理学的に関連する質問をするための手段を提供します。

どのように文化、レコードとマイクロ電極アレイで神経ネットワークを刺激する（多国間環境協定）へ

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

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How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

54.5K ビュー.  Emory University School of Medicine. このプロトコルは、セットアップの世話から、記録と電気的に多国間での文化を刺激するために必要な情報を提供します。試験管内でネットワークは、脳機能と機能障害のより良い理解につながるネットワークと細胞レベルでの生理学的に関連する質問をするための手段を提供します。

ビデオ: How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

神経毒性の高いコンテンツ分析のための神経とアストロサイト共培養アッセイ

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

生物学

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

チック EX卵文化と EX卵 CAMアッセイ：それが実際にどのように動く

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

細菌機能ネットワークとPathwaysでのマッピング<em&gt;大腸菌</em&gt;合成遺伝子配列を用いて

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

多重ケモスタットアレイの設計および使用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

外酸生産の測定と解析は、解糖速度を決定するために

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Techniques to Induce and Quantify Cellular Senescence

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed cellular senescence. Accumulation of senescent cells occurs with organismal aging and through continual culturing in vitro. Senescent cells influence many biological processes, including embryonic development, tissue repair and regeneration, tumor suppression, and aging. Hallmarks of senescent cells include, but are not limited to, increased senescence-associated &#946;-galactosidase activity (SA-&#946;-gal); p16INK4A, p53, and p21 levels; higher levels of DNA damage, including &#947;-H2AX; the formation of Senescence-associated Heterochromatin Foci (SAHF); and the acquisition of a Senescence-associated Secretory Phenotype (SASP), a phenomenon characterized by the secretion of a number of pro-inflammatory cytokines and signaling molecules. Here, we describe protocols for both replicative and DNA damage-induced senescence in cultured cells. In addition, we highlight techniques to monitor the senescent phenotype using several senescence-associated markers, including SA-&#946;-gal, &#947;-H2AX and SAHF staining, and to quantify protein and mRNA levels of cell cycle regulators and SASP factors. These methods can be applied to the assessment of senescence in various models and tissues.

Cellular senescence, the irreversible state of cell-cycle arrest, can be induced by various cellular stresses. Here, we describe protocols to induce cellular senescence and methods to assess markers of senescence.

誘導および定量化細胞老化するテクニック

In response to cellular stress or damage, proliferating cells can induce a specific program that initiates a state of long-term cell-cycle arrest, termed ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+過渡現象とL型カルシウム電流の同時測定のための高品質マウス心房および心室筋細胞の分離

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Divergence of Root Microbiota in Different Habitats based on Weighted Correlation Networks

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, which can effectively explore the interaction relationship or co-occurrence model of different microbial species in different environments. The purpose of this manuscript is to provide details on how to use the weighted correlation network algorithm to analyze different co-occurrence networks that may occur in microbial communities due to different ecological environments. All analysis of the experiment is performed in the WGCNA package. WGCNA is an R package for weighted correlation network analysis. The experimental data used to demonstrate these methods were microbial community data from the NCBI (National Center for Biotechnology Information) database for three niches of the rice (Oryza sativa) root system. We used the weighted correlation network algorithm to construct co-abundance networks of microbial community in each of the three niches. Then, differential co-abundance networks among endosphere, rhizoplane and rhizosphere soil were identified. In addition, the core genera in network were obtained by the "WGCNA" package, which plays an important regulated role in network functions. These methods enable researchers to analyze the response of microbial network to environmental disturbance and verify different microbial ecological response theories. The results of these methods show that the significant differential microbial networks identified in the endosphere, rhizoplane and rhizosphere soil of rice.

Network analysis was applied to evaluate the association of various ecological microbial communities, such as soil, water and rhizosphere. Presented here is a protocol on how to use the WGCNA algorithm to analyze different co-occurrence networks that may occur in the microbial communities due to different ecological environments.

重み付け相関ネットワークに基づく異なる生息地における根内微生物叢の発散

The root microbiome plays an important role in plant growth and environmental adaptation. Network analysis is an important tool for studying communities, ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

成人筋幹細胞機能評価のための単一筋繊維培養アッセイ Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...