この作品は、NIH R01 GM47607、CA163296およびCETにNSF 1334493によってサポートされています。私たちは、原稿の重要な読書のためのターナーラボのメンバーに感謝します。このレポートに表示されるすべてのデータは、駆虫剤ら。2014年11から許可を得て再現されました。

Durotaxisチェンバースの調製<ol><li> 1つの6ウェル組織培養プレートを調製するために、50 mlのコニカルチューブとのバランスを風袋引き。 50 mlチューブ中のPDMSベース溶液約10グラムを秤量（溶液は非常に粘性があります）。 </li><li> 90：1の基板（〜1キロパスカルのコンプライアンス）、必要に応じて架橋剤溶液の正確な量を決定するために90によってチューブ内のPDMS塩基溶液の測定された重量を分割。同じチューブにPDMSの架橋剤溶液の計算された量を追加します。 例：10グラム/ 90 = 0.11グラム。 PDMSベースのソリューションへのPDMS架橋剤溶液の0.11グラムを追加します。 注：ベースと架橋剤溶液は、両方のPDMSキットで提供されています。 </li><li>精力的に小さなへらを使用して室温で5分間、PDMSベース/架橋剤混合物を混ぜます。この段階で、混合物を多数の気泡を含むであろう。 </li><li> BUBを除去するために、室温で50×gで5分間、卓上遠心機でPDMS基板を遠心BLES。残っている場合は、再び5分、遠心分離後に泡。 </li><li> 90 mlのピ​​ペット1：組織培養の各ウェルに1 PDMS基板を6ウェルプレートを処理しました。 PDMS中に存在する任意の残存する気泡が21 G針を使用してそれらをポップすることにより、この段階で除去することができます。 PDMSは、ウェル中の30分間の広がりを許可します。 </li><li>ボイル12ミリメートルのガラス＃5分間蒸留水で1カバースリップ。 2回繰り返し、蒸留水にカバースリップを格納します。 </li><li>静かにPDMS上にカバーガラスをドロップし、PDMS溶液にカバースリップの片側をタッチすることで、組織培養プレートの各ウェルに1、乾燥したカバーガラスを配置します。カバーガラスが安定するように、PDMSはカバーガラスの縁の上に侵入し始めますが、それを完全にカバーしています。これは、（ 図1A、B参照）硬化後、PDMSとガラスの間のインターフェイスを生成します。 </li><li> （硬化）PDMSを硬化させる16時間オーブンで70℃でプレートをインキュベートします。 PLATを配置細胞培養フードとUVの電子は、10分間滅菌します。 注：これは、使用の数日以内にプレートを作るのがベストです。しかし、プレートは任意のバッファなしパラフィルムで包み、品質の顕著な低下させることなく2週間まで4℃で保存することができます。 </li></ol> 2.細胞播種注：siRNA媒介ノックダウンの効果を研究する場合は、製造元の指示や選択の細胞型に最適化されたプロトコルを使用してノックダウンを行います。 <ol><li>各コートは、37°C​​で1時間、カルシウムおよびマグネシウムを含まないPBS中10μg/ mlのフィブロネクチン1mlのチャンバdurotaxis。あるいは、フィブロネクチン、4℃で16時間、PBS中10μg/ mlのフィブロネクチンでPDMSをインキュベートすることによって、分析前に一日適用することができます。全面をPBS溶液中のフィブロネクチンに沈めていることを確認します。 </li><li>熱変性1％BSAを準備します。 0.5グラムのBSAを秤量し、PBS 50ml中に溶解します。フィルターは、12分間80℃で0.22μmのフィルターと熱を通して溶液を滅菌します。 注：前に4℃での細胞播種と店に一日、この溶液を調製します。 </li><li>フィブロネクチン溶液を吸引し、PBSで3回洗浄します。各ウェルにPBS中の熱変性BSAを1ml加え、室温で30分間インキュベートします。 </li><li> durotaxis室が熱変性BSAでブロックしている一方でトリプシン処理し、選択の細胞をカウントします。 </li><li>プレートの選択の特定の細胞型に必要なメディアを使用してdurotaxis室の各ウェルに2ミリリットルの容量で、1×10 5細胞 、。細胞が付着し、5％CO 2で37℃の加湿インキュベーター中で4時間、基板上に塗布することを許可します。 注：U2OS細胞を日常的に2mMのL-グルタミン、1mMピルビン酸ナトリウム、および10 IU / mlのペニシリン、を10μg/ mlのストレプトマイシンを補充した、10％FBSを含むDMEM中で維持されています。 注：この例で使用される細胞数は、のために最適化されU2OS細胞。他の細胞型のための最適化が必要とされ得ます。この密度は、細胞を他の細胞との相互作用の重要なことなく移行するための十分なスペースを提供します。 </li></ol> 3.ライブセルイメージング<ol><li> 10倍の対物レンズとの位相差を使用して、倒立顕微鏡でライブセルイメージングを実行します。顕微鏡は、長期的画像化の間に37℃の温度を制御し、5％CO 2を可能に囲まれた環境、加湿チャンバーを装着しなければなりません。 </li><li>細胞は約3.5時間に広がった後、顕微鏡室にプレートを組み立てます。サンプル（s）は30分間チャンバー内で平衡化することを許可します。 </li><li>利用可能な場合、顕微鏡に訪問し、自動化、マルチポイントを設定します。 90との間のインターフェイスに焦点を当て：1 PDMSとカバーガラスとdurotaxis室あたり40点の平均とすべてのインターフェイスの周りに画像にポイントを選択します。画像セルまでの16時間ごとに10分。 注：再興味のある祇園は、カバーガラスのエッジとPDMSとカバーガラスとの間の実際のインタフェースに対応する内側のラインに対応する外側のラインで、二行として表示されます。 図1Bを参照してください 。 </li></ol> 4.データ解析<ol><li> 表1のように 、スプレッドシートを生成します。 </li><li>生成された各ムービーからガラス表面およびその逆に、PDMSから交差イベントの数をカウントします。 Excelスプレッドシート内の適切な列に交差するイベントの数を記録します。 注：交差イベントがいずれかの方向にPDMSとガラスの間の境界を通過する細胞の核として定義されます。 </li><li>複数の交差を定量化するために、細胞を界面を越えた回数をカウントします。その数は、細胞は、映画の最後に位置された基板に対応するExcelの列に記録する必要があります。トンのインターフェイスを横断するセル毎に解析を繰り返し彼の映画。撮像中に視野の外に移動する細胞を除外します。 注：これは、細胞は、フィブロネクチンでコーティングされたPDMSとカバーガラスの表面に均等に付着することができることを、実験の開始時に細胞計数によって、検証することも重要です。 </li><li> （ すなわち、durotaxisを受けた）ガラス表面にPDMSから遊走した細胞の割合を計算します。ソフトからハードまでのイベントと交差イベントの合計数で、ハードと除算に終了した複数の交差事象を通過する数を追加します。 </li><li>交差の総数で複数の交差の数を分割して複​​数の交差の割合を計算します。 </li></ol>

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The authors have no conflicts to disclose.

ここで我々は、細胞の移行にdurotaxisを研究するための単純なアッセイを説明します。このアッセイの主な強みは、PDMSを用いdurotaxisチャンバを調製する容易さです。基板の剛性を容易にアッセイにおける様々な剛性の研究を可能にするために、架橋剤にPDMSベース溶液の比率を変えることによって操作することができます。しかし、システムの潜在的な制限は、より洗練されたシステム4</sup...

細胞外マトリックス（ECM）は、コラーゲン、フィブロネクチンおよびラミニンを含む構造と架橋タンパク質の複雑な配列で構成されています。それは十分にECMは細胞組織のための重要な構造的支持を提供することが確立されているが、細胞が活発に細胞生存、分化および細胞遊走を含む多様な細胞プロセスを調節するために、それらのECM環境の物理的変化に応答することを示す証拠が増えてきています。硬い（〜25キロパスカル）の基板は骨形成分化1を促進しながら、例えば、ECMの剛性の違いは、ソフト基板（〜1キロパスカル）神経性系統を促進して、異なる系統に向けて、間葉系幹細胞を駆動することができます。同様に、間質マトリックスの剛性の増加は、周囲の組織2,3に乳腺上皮細胞の腫瘍形成および浸潤を促進することが示されています。 このmechanosignの特に興味深い点細胞がよりリジッド基板4,5の方に優先的に移行するdurotaxis、として知られているプロセスでの活動結果をaling。細胞は常に、ECMに結合するインテグリン受容体を介して、それらの細胞外環境の物理的特性を感知します。これは、順番に、接着斑または焦点コンタクト6,7として公知の接着剤構造の形成を駆動するために、それらの細胞質ドメインに、多数の構造的およびシグナル伝達タンパク質の蓄積を促進します。 インテグリンは全く固有の酵素活性を有していないので、信号がその環境変化〜8セルの応答を調整するために、これらのアクセサリータンパク質を介して、ECMから中継されます。従って、mechanosignalingとdurotaxisの調節に関与する重要なタンパク質の同定および特徴付けは、研究の重要な領域です。 様々なモデル系は 、in vitroでdurotaxisを研究するために開発されてきたが、ほとんどが持っていますコラーゲンコートポリアクリルアミド基材4を用いました。しかし、ポリアクリルアミド基質の製造は技術的に困難であることができ、これらのアッセイで使用されるコラーゲンは、 基板 9に化学的に架橋する必要があります。ポリジメチルシロキサン（PDMS）基板、ポリアクリルアミド基板10に匹敵する機械的特性を示すことが示されています。しかし、PDMS基板は、単に架橋剤を塩基の比を混合することによって調製され、これらの基板は、このようにPDMS細胞挙動に対する剛性​​の影響を研究するための容易なツールを作製する、化学的架橋を必要とせずに、ECMタンパク質でコーティングすることができます。ここで、我々はソフトPDMS基板は、硬質ガラスカバースリップで一体化された単純なdurotaxis室を準備する方法について説明します。 アッセイは、以下に概説するように、durotaxisを研究するために迅速かつ簡単な方法を提供します。この研究のために、我々は、siRNA媒介と組み合わせ、人間のU2OS骨肉腫細胞を使用しましたdurotaxis 11でこの焦点接着タンパク質の役割を研究するcdGAPのノックダウン。重要なことに、このプロトコルは、容易に個々の要件に適合させることができます。他の細胞型は、U2OS細胞に置換されてもよく、任意のタンパク質をノックダウンまたはdurotaxis中の細胞の挙動に影響を決定するために過剰発現することができます。さらに、このプロトコルは、FRAPまたはFRETの手法を使用してダイナミックス及び挙動を分析するためにタグ付けされた蛍光タンパク質を組み込むように適合されてもよいです。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
		</tr>
		<tr>
			<td>#1 Cover glass 12mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-82</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro</td>
			<td>15-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Fisher Scientific</td>
			<td>BP356-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>610077</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>21600-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Celltreat</td>
			<td>229456</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simplified system for evaluating cell mechanosensing and durotaxis in vitro

細胞外マトリックスの組成及び機械的特性は、組織型間で高度に可変です。この結合組織の間質の多様性に大きく影響を与える細胞の挙動細胞増殖、分化、接着シグナリングおよび方向性のある移動を含む正常および病理学的過程を調節するために。この点において、特定の細胞型の生来の能力がdurotaxisと呼ばれる堅い、以下に準拠マトリクス基板に向かって移動します。この現象は、胚発生、創傷修復及び癌細胞浸潤の間に重要な役割を果たしています。ここでは、ポリジメチルシロキサン（PDMS）基板を用いて、in vitroで 、durotaxisを研究するための簡単なアッセイを説明します。室durotaxis記載の製造は比較的柔らかいPDMSゲルと硬質カバーガラスとの間に剛性のインターフェースを作成します。提供された例では、CDC42 / Rac1のGTPアーゼ活性化proteの役割を実証するために、これらのdurotaxis室を使用していましたcdGAP、で、mechanosensingと人間のU2OS骨肉腫細胞におけるdurotaxis規制インチこのアッセイは、他の細胞型および/またはmechanosignalingとdurotaxisにおけるそれぞれの役割を探求するための利益の他のタンパク質のノックダウンに容易に適合可能です。

durotaxisチャンバの概略図は 、図1Aに示されています。ソフトPDMS基板を、（90：ソリューションを架橋するために、PDMSベースの1混合物）を6ウェル皿に広げ、カバーガラスがそれによってインターフェースを作成し、部分的にカバーガラスの上面を覆うPDMSの上に配置されます異なるコンプライアンスの2枚の基板の間。ソフトPDMS基板の剛性は、ガラスの剛性は約1 GPaで1~2で...

Watch this Scientific Journal Video about A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro at JoVE.com

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

セルMechanosensingとDurotaxisを評価するための簡略化したシステム<em&gt;インビトロ</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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Research

JoVE Journal

Biology

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

8.1K ビュー.  SUNY Upstate Medical University. Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

ビデオ: A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

エンハンサー配列を評価するためのモザイクのゼブラフィッシュのトランスジェネシス

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

生物学

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

植物では無細胞系でプロテアソームによる分解をアッセイ

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮細胞接着性の定量<em&gt;インビトロ</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

統合欠陥レンチウイルスベクターにパッケージ化された転写調節睡眠美容システムによって効率的な体外転位法

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

細胞運動と関連するシグナル伝達事象の機械的制御を評価するための単細胞デュロタシスアッセイ

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

フィーディングアッセイによる Helicoverpa armigera に対する植物化学的影響の評価

Developing a Feeding Assay System for Evaluating the Insecticidal Effect of Phytochemicals on Helicoverpa armigera

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and agricultural productivity. In response, plants produce several phytochemicals that negatively impact the insect's growth and survival. This protocol demonstrates an obligate feeding assay method to evaluate the effect of a phytochemical (quercetin) on insect growth, development, and survival. Under controlled conditions, the neonates were maintained until the second instar on a pre-defined artificial diet. These second-instar larvae were allowed to feed on a control and quercetin-containing artificial diet for 10 days. The insects' body weight, developmental stage, frass weight, and mortality were recorded on alternate days. The change in body weight, the difference in feeding pattern, and developmental phenotypes were evaluated throughout the assay time. The described obligatory feeding assay simulates a natural mode of ingestion and can be scaled up to a large number of insects. It permits one to analyze phytochemicals' effect on the growth dynamics, developmental transition, and overall fitness of H. armigera. Furthermore, this setup can also be utilized to evaluate alterations in nutritional parameters and digestive physiology processes. This article provides a detailed methodology for feeding assay systems, which may have applications in toxicological studies, insecticidal molecule screening, and understanding chemical effects in plant-insect interactions.

著者スポットライト:害虫管理のための給餌アッセイの開発と課題 

This protocol describes the obligate feeding assay to evaluate the potentially toxic effect of a phytochemical on the lepidopteran insect larvae. This is a highly scalable insect bioassay, easy to optimize the sublethal and lethal dose, deterrent activity, and physiological effect. This could be used for screening eco-friendly insecticides.

Helicoverpa armigeraに対するファイトケミカルの殺虫効果を評価するための摂食アッセイシステムの開発

Helicoverpa armigera, a lepidopteran insect, is a polyphagous pest with a worldwide distribution. This herbivorous insect is a threat to plants and ...