このプロジェクトは、公衆衛生のイリノイ州省と国立科学財団（DBI - 0852416）により賄われていた。

1。デバイスの機能<ol><li>低酸素挿入装置は、標準の6ウェルプレートに巣その6柱を含んでいます。ガスは、柱の基部に微小流体ネットワークを介して、柱に流れ込み、およびデバイスの背面に流れ出します。マイクロチャネルの底壁を構成する柱の基部にマイクロガスと培地との間で酸素の拡散を可能にする厚さ100μmのガス透過性PDMS膜です。したがって、デバイスが所望の値に向かって、メディアの酸素濃度を駆動する濃度勾配を確立することによって動作します。 </li><li>このデバイスは、低酸素室と他の細胞の酸素化のシステムに比べ多くの利点提供しています：1）、2を速く時間的制御を可能にする酸素の拡散経路を最小限に）柱マイクロチャネルの設計上のような依存酸素化における空間制御、3を強化）を有する小さな研究室の設置面積と実験効率を高めるためにより高いスループット、および4）専門的な知識や機器を必要とせずに一般的な細胞培養ツール（例えば、マルチウェルプレート）に適応します。 </li></ol> 2。デバイス製造<ol><li>第一の柱の配列は以前に加工されたDelranの金型にPDMSで成形されたレプリカです。 </li><li>各ピラーの下部にマイクロチャネル次にガスは、標準のSU - 8フォトリソグラフィーとPDMSレプリカモールドを用いて製造されています。 </li><li>ガス透過性膜は、所望の厚さを達成するために、シリコンウエハ上にPDMSの定義紡糸により製造されています。この例では、10秒間に500回転してから30秒間、900 rpmの回転により作製された厚さ100μmメンブレンを使用してください。 </li><li>すべてのコンポーネントは、ハンドヘルドプラズマ装置（モデルBD - 20、エレクトロテクニックプロダクツ）と酸素プラズマ処理の後に一緒に結合されています。 </li></ol> 3。デバイスのセットアップ<ol><li>静かに泡を避けるために確認して、プレートにデバイスを挿入します。挿入中にデバイスを釣りには片側に気泡を追い出すのに役立ちます。実際の細胞ベースの実験については、このステップは、無菌層流フードの内部で行う必要があります。デバイスが挿入されると、汚染の可能性を大幅に削減され、アセンブリが​​非無菌環境でのインキュベーターに繰り越すことができるので。 </li><li>装置の入口側と出口側に原料ガスのタンクからチューブを接続します。細胞ベースの実験のために、培養インキュベーターは、チューブのエントリを許可する穴があるはずですし、チューブは、デバイスを運ぶし、インキュベータにそれを配置した後に接続する必要があります。基盤となる細胞を破砕するのに十分なPDMSを変形させることができる装置、に過剰な圧力を置かないように注意してください。 </li><li>精密フローレギュレータがデバイスのオーバーフローを避けるためにタンクトップレギュレータを開く前に閉じていることを確認してください。ガスの流れを開始します。所望の流量（50〜100 mL /分）にゆっくりと開いている精密フローレギュレータ。システムは、流量を変えること、平衡化しながら、圧力降下が変化するので、次の時間にわたって密接に価値を見て、必要に応じて調整を行います。メディアにおける気泡の形成を回避するために、より高い流速を持つ最初の15分間の平衡期間の後に10から20の間mL / minに流量を減少させる。 </li><li>目的の実験期間中、ガスの流れを停止した後、プレートを取り外し、およびプロセスの細胞に応じて（例えば等、数え、染色、溶解）。 </li></ol> 4。デバイスの検証<ol><li> 校正 <ol><li>蛍光酸素の位置の数と場所を選択 酸素の測定に使用される（FOXY）センサーのスライド（検証の要件に応じて）。スライドは、酸素によって消光される蛍光ルテニウム色素のコーティングが含まれています。 </li><li> 0、10を直接スライド公開、およびガスのタンクから21％の酸素と適切な平衡化のため5分後に画像をキャプチャ。 </li><li>蛍光強度に依存するように各位置とプロットの酸素濃度の平均画像の輝度をエクスポートします。 </li><li> 0〜10％のラインと10から21パーセントのラインに線形の曲線をフィッティングすることにより検量線を生成します。 </li></ol></li><li> 異質 <ol><li>定義された間隔（例えば各1 mm）でのチャネルの幅をまたがる複数のポイントを確立する。間隔に応じて、画像の重複があるかもしれないことに注意してください。 </li><li>デバイスを介しても表面で酸素濃度を測定する</li></ol></li><li> 平衡 <ol><li>酸素濃度を測定するためにFOXYスライド上の3つの点を選択してください。 </li><li>すぐに周囲の酸素濃度での最初の画像をキャプチャした後、デバイスへのガスの流れを開始するために精密なフローレギュレータを開きます。酸素concentrの期間と程度を評価するために適切な間隔で画像をキャプチャエーションの平衡（30分間毎に10秒など）。 </li></ol></li></ol> 5。アプリケーション<ol><li> 創傷治癒 <ol><li>実験前日、ウェル中の気泡の形成を抑制減らすために無血清培地に滅菌PDMSインサートを浸す。 </li><li> 6ウェルプレートでコンフルエントに100％の培養細胞。 </li><li>傷をシミュレートするために、P200のピペットチップを用いて単層でストレート傷を作成します。 </li><li>細胞の培地を吸引除去する、5mLのメディアですすいでください、そして再び吸引。細胞の単層を乱さないことが重要です。 </li><li>リフィル細胞増殖を減少させるために4 mLの無血清培地をウェルの中。 </li><li>場所は、井戸に挿入し、対応する酸素の濃度を各ウェルに接続します。 </li><li> 37℃加熱ステージ上のインサートを持つ場所を6ウェルプレート℃に</li><li>目的の間隔と継続時間の合計で細胞のタイムラプス画像をキャプチャします。 MATLABのT_Scratch、傷の測定アルゴリズムは、治癒表面積を分析するために使用することができます。 </li></ol></li></ol> 6。代表的な結果<ol><li> デバイスの検証 低酸素挿入装置は、0.5％の酸素に安定するまで2分未満を必要とする、酸素の平衡時間と範囲の面で低酸素室と比較して大きな改善を示す。デバイスの膜ウェルボトムギャップサイズは大きくギャップのサイズは、定常状態の酸素濃度の値に到達するより多くの時間を必要とすると、平衡の効率を左右する重要な要因だった。また、このデバイスは、複数の条件の形成を可能にする、単一のウェル内の空間的な酸素化を制御するための多くを許可し、さらにウェルの表面全体に周期的な酸素のプロファイルを生成する。 </li><li> 創傷治癒 細胞単層は、10％または21％の酸素にさらされ、創傷面の面積は、時間をかけて分析した。 21％の酸素にさらされた傷は、最も遅いと10％の最速を閉じた。図1は、17時間の経過とともに傷の画像を示す。図5のグラフは、実験期間中の酸素濃度の両方のためのオープンな創傷面積の割合を示しています。 </li></ol> <a href="https://www.jove.com/files/ftp_upload/1695/1695fig1.jpg"><img src="/files/ftp_upload/1695/1695fig1tmb.jpg" alt="図1" border="0" /></a> 図1の回路とデバイスの機能を説明する図。酸素挿入装置はレプリカは（マイクロ流体ネットワークとインサート足場）成形、従来のフォトリソグラフィ（マイクロ流体ネットワーク）で作製し、PDMS（ガス透過性膜）の紡糸定義されています。 A）酸素のデバイスは、6ウェルプレートにネスト。 B）24および96ウェル柱の配列の例。柱のC）の断面概略図。酸素は、入口を通って装置内に流入し柱の下部に微小流体ネットワークを通過します。酸素は自由に柱の下部にガス透過性のPDMS膜を横切って拡散し、培地中に溶解することができる。 D）平衡の研究のための結合されたガラスのポストで、上からのシングルチャネルの柱の様々な機能を示す顕微鏡写真。 <a href="https://www.jove.com/files/ftp_upload/1695/1695fig2.jpg"><img src="/files/ftp_upload/1695/1695fig2tmb.jpg" alt="図2" border="0" /></a> 図2の酸素センサーとデバイスの検証。各ウェル内の酸素分圧は、平面ルテニウムの酸素センサを用いて特徴付けられた。すべての酸素の混合物は、メディアのバッファリングのためにバランスの窒素および5％CO 2を含んでいた 。 A）プロットは平衡時間および有効性を、後の高さの影響を示すため、細胞膜と細胞間の酸素の拡散距離。ハイツは、装置の底部にバインドされたカットグラスのポストによって設立された。すべての3つのポストのサイズは非常に低酸素室で改善平衡化時間を得る。時間は対数スケール上にあることに注意してください。 B）プロットは、0.2 mmギャップデバイスの急速な酸素の平衡応答時間を描いた。 C）マルチポジションのラインスキャンは、デバイスによって導入された酸素濃度の均一性を確保するためのマイクロチャネルでも越えて行われた。グラフは、0％、10％、および10分間21％の酸素を注入した後に測定した酸素濃度を示しています。 D）デバイスは、効果的に5日間にわたる10％の酸素を維持しています。 <a href="https://www.jove.com/files/ftp_upload/1695/1695fig3.jpg"><img src="/files/ftp_upload/1695/1695fig3tmb.jpg" alt="図3" border="0" /></a> 図3。複雑な酸素マイクロチャネルの設計と実験。 A）デュアル条件マイクロチャネル設定は、安定した0％、14日以上21％酸素のプロファイルを得られます。 B）番目にわたって延びる500μmの幅のマイクロチャネルの櫛歯と巻線パターンは、巡回酸素プロファイルのEピラー結果。マイクロチャネルアライメントが困難だったので、データは1つだけ代表的な裁判を描いていることに注意してください。 <a href="https://www.jove.com/files/ftp_upload/1695/1695fig5.jpg"><img src="/files/ftp_upload/1695/1695fig5tmb.jpg" alt="図4" border="0" /></a> 図4。創傷閉鎖0、7、および初期傷後17時間のタイムラプス画像。細胞は実験期間を通じて21％の酸素を配信されていました。 <a href="https://www.jove.com/files/ftp_upload/1695/1695fig6.jpg"><img src="/files/ftp_upload/1695/1695fig6tmb.jpg" alt="図5" border="0" /></a> 図5。スクラッチアッセイにおける創傷治癒の速度での酸素濃度の影響。

デバイスは、標準のSU - 8フォトリソグラフィー、レプリカの成形、および定義されたスピンとポリジメチルシロキサンから完全に作られたことにより製造されています。ガスは、所望の平衡酸素濃度に向けてシステムを駆動する、マイクロチャネルの柱と培地の間に濃度勾配を確立するためにデバイスに導入される。デバイスは、効果的にも内部に時間的および空間的な酸素化を調節するだけでなく、適切な細胞挙動を調節することが示されている。酸素化の空間パターン形成が柱の基部でマイクロチャネルによって定義され、デザインの様々なフォトマスクを作り上げるに実施することができるので。さらに、井戸の気相への所望のガスの注入は、平衡時間と低酸素の程度の向上が期待される。マイクロ流体混合ネットワークは、少数の株式のガスのタンクから新規な混合ガスを生成する手段を提供するデバイスに適合させることができる。最後に、メディア交換のためのメカニズムは、細胞が応答する可能性があるからマルチウェルプレート、からデバイスの取り外しの必要性を削除してしまうことになります。 デバイスは、 インビトロまたは酸素濃度の制御を必要とするex vivoでの実験で任意のアプリケーションを持っています。酸素は、シグナル伝達経路の大部分に影響を与える重要な生理学的変数であるため、恩恵を受ける研究の分野は、研究者の創造性によって制限されます。酸素濃度の強​​化された時間的な制御の恩恵を受ける一部のフィールドは、他の多くの間で癌の転移、睡眠時無呼吸症候群、および心臓の虚血再灌流障害を、含まれています。例えば、間欠的低酸素症は、連続的な低酸素症および正常酸素の相対metastastis関連遺伝子の数を上方制御する、より多くの浸潤癌と相関している。空間的な制御は、酸素勾配が開発、肝臓の帯状分布、薬物毒性、および幹細胞のニッチで重要であるため、も重要です。この記事で紹介した装置は、小さなラボフットプリント、比較的単純な運用上の要件、および細胞への酸素曝露上はるかに優れた制御システムを提供することにより、研究の多くの分野の利益になる。

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<td>PDMS-Sylgard 184</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>Gas</td>
<td>&nbsp;</td>
<td>Airgas</td>
<td>Custom mixes</td>
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fabrication and operation of an oxygen insert for adherent cellular cultures 

酸素は多くの細胞経路の重要なモジュレーターであるが、現在のデバイスが良ければ<em&gt; in vitroで</em&gt;酸素の変調は、生物医学研究のニーズを満たすことができない。低酸素室は、標準的な培養容器内の酸素化を制御するシンプルなシステムを提供していますが、生理現象のさまざまな研究に、そのアプリケーションを防止する、細胞表面での酸素濃度を正確に時間的空間的制御を欠いている。他のシステムでは、低酸素室に改良が、平均的な研究者のためのそれらを威圧すること、それらの操作のための専門知識と機器を必要としている。マルチウェルプレートのための微細加工インサートは、より効果的に発見より良いモデル生理現象に時間と空間の酸素濃度を制御するために開発されている<em&gt; in vivoで</em&gt;。プラットフォームは、標準のマルチウェルプレートに巣そのポリジメチルシロキサンのインサートで構成されており、付着細胞への酸素供給を調節することを目的としたガス透過膜とパッシブマイクロ流体ガスのネットワークとして機能します。このデバイスは使いやすく、プラットフォームに必要な酸素濃度を導入する圧力を提供するガスボンベに接続されています。製造は、シリコンウェハ上にスピン標準のSU - 8フォトリソグラフィー、レプリカの成形、および定義されたPDMSの組み合わせが含まれます。デバイスのコンポーネントは、ハンドヘルド型プラズマシステムを使用して表面処理後に接着されている。検証は、平面蛍光酸素センサーで実現されます。平衡時間は分のオーダーであり、酸素のプロファイルの多種多様なデバイスの設計に基づいて達成することができる、そのような本研究で達成された周期的なプロファイルとして、さらには酸素勾配が見られるものを模倣する<em&gt; in vivoで</em&gt;。デバイスは、機能を失うことなく一般的な方法を用いて細胞培養のために滅菌することができる。勉強へのデバイスの適用性<em&gt; in vitroで</em&gt;創傷治癒の応答が実証されます。

Watch this Scientific Journal Video about Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures at JoVE.com

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

6ウェルプレートでの空間的、時間的な酸素化のコントロールが強化さを提供するアドオンプラットフォームの作製と検証。デバイスは、文化のシステムの数に適応可能であり、創傷治癒に及ぼす酸素の影響を調査するために使用することができます。

接着細胞培養用の酸素挿入の作製と動作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

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JoVE Journal

Biology

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

9.6K ビュー.  University of Illinois. 6ウェルプレートでの空間的、時間的な酸素化のコントロールが強化さを提供するアドオンプラットフォームの作製と検証。デバイスは、文化のシステムの数に適応可能であり、創傷治癒に及ぼす酸素の影響を調査するために使用することができます。

ビデオ: Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

大規模な挿入環境ゲノムライブラリーの製造

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

生物学

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

接着および非接着性の哺乳動物細胞の in situ細胞下分画で

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

クローン原性アッセイ：付着細胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

三次元蛍光画像から細胞形態の変化を定量分析ツール

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

頭蓋間葉の細胞挙動を評価するための外植片アッセイ

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

、検出し可視化し、アメーバモデル系で活性酸素種の生成を定量

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt to make straightforward the very complex relationships between the several cellular subtypes that compose multicellular organisms, in vitro techniques have been developed to help researchers acquire a detailed understanding of single cell populations. One of these techniques uses inserts with a permeable membrane allowing secreted soluble factors to diffuse. Thus, a population of cells grown in inserts can be co-cultured in a well or dish containing a different cell type for evaluating cellular changes following paracrine signaling in the absence of cell-cell contact. Such insert co-culture systems offer various advantages over other co-culture techniques, namely bidirectional signaling, conserved cell polarity and population-specific detection of cellular changes. In addition to being utilized in the field of inflammation, cancer, angiogenesis and differentiation, these co-culture systems are of prime importance in the study of the intricate relationships that exist between the different cellular subtypes present in the central nervous system, particularly in the context of neuroinflammation. This article offers general methodological guidelines in order to set up an experiment in order to evaluating cellular changes mediated by secreted soluble factors using an insert co-culture system. Moreover, a specific protocol to measure the neuroinflammatory effects of cytokines secreted by lipopolysaccharide-activated N9 microglia on neuronal PC12 cells will be detailed, offering a concrete understanding of insert co-culture methodology.

In multicellular organisms, secreted soluble factors elicit responses from different cell types as a result of paracrine signaling. Insert co-culture systems offer a simple way to assess the changes mediated by secreted soluble factors in the absence of cell-cell contact.

細胞間接触の不在で2つの細胞タイプの挿入共培養システムの開発

The role of secreted soluble factors in the modification of cellular responses is a recurrent theme in the study of all tissues and systems. In an attempt ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

カポジ肉腫ヘルペス ウイルスによる細胞変換を勉強するための in Vitroモデル

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled conditions.

化学発光アッセイによるイネの免疫応答における活性酸素種産生のリアルタイム検出

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen ...

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

簡単な2Dおよび3D細胞培養を可能にするように設計された革新的な3Dプリントインサート

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...