この研究は、米国立衛生研究所の国立癌研究所の学内研究プログラムによって部分的にサポートされており、NCIによってUA5CA152907を付与した。

血管新生の可能性をテストするために調節された媒体の1集<ol><li>ネイティブまたは低血清培地中での血管新生または抗血管新生の可能性について試験されるべき一次または不死化細胞を増殖し、馴化培地を収集します。 <ol><li>あるいは、使用がすぐに馴化培地、または等分し、数ヶ月間-80℃で保存した。ネガティブコントロールとしてnonconditioned天然又は低血清培地を使用し、ポジティブコントロールとしての非馴化完全な増殖培地（10％のFBS、または適切な濃度）を使用します。あるいは、血管新生の潜在的な刺激物質をテストするために、関心のある特定のタンパク質の既知の濃度の成長因子を欠いているサプリメントチャコール処理メディアだけでは、成長因子を含まない培地と比較します。 </li></ol></li></ol>アッセイの前に内皮細胞の調製<ol><li>アッセイの2日前に、新しいT-75フラスコ内に通路3B-11細胞。細胞の継代数は重要です。細胞は第二と第六の通路の間にある場合、このアッセイは、最も効果的に機能します。アッセイは、内皮細胞は第二の前または第十経過した後に使用されている場合にはうまく機能しません。 </li><li> 10％FBS、2mMのL-グルタミン、1mMピルビン酸ナトリウム、100 U / mlペニシリン及び100μg/ mlストレプトマイシンを補充したDMEM中で24時間細胞を成長させる。 </li><li>以下のようにアッセイの1日前に、血清は3B-11細胞を飢え： <ol><li> 3B-11細胞から培地を吸引。 </li><li> DMEMの追加低減血清培地は、0.2％のFBS、2mMのL-グルタミン、1mMのピルビン酸ナトリウム、100 U / mlペニシリン及び100μg/ mlストレプトマイシンを補充した。 </li><li>さらに24時間細胞を成長させる。 </li></ol></li></ol>アッセイの前に試薬の調製注：BME濃度は、多くの依存性の高い変数である。濃度がそのためBMEメーカーが保証するために購入前に連絡する必要があり、10mg未満/ mlである場合にはBMEはうまく動作しません十分に集中し、多くの買収。 <ol><li>デフロストは、冷蔵庫に成長因子BMEを減少させた。時暖かいBMEが固化するので、使用前に風邪に保つ必要があります。 BMEは、数週間の冷蔵保存、あるいは等分し、-80℃で凍結することができる。 </li><li>成長因子低減BMEは、従来のBMEより好ましい。 BMEにおける血管新生因子の欠如は、簡単に条件培地からの要因が管形成に与える影響を観察するようになります。 </li><li>解凍メディアがテストされる。 </li></ol>アッセイの直前に、内皮細胞の4。準備<ol><li>ウォッシュDPBS中3B-11細胞。 <ol><li>蛍光または共焦点顕微鏡による可視化が所望される場合、3B-11細胞を洗浄する前に、2μg/ mlの最終濃度で培地中の内皮細胞へのカルセインAMを加える。アッセイの開始前に37℃および5％CO 2の暗所でカルセイン標識された細胞を配置する。在庫カルセインを溶解DMSOおよびカルセインAMは、一度DMSOの最終濃度が0.1％を超えるべきではない培地で希釈する。細胞（上記のステップ4.1）から30〜45分間洗浄カルセインAMの後、実験は以下の通りに進みます。 </li></ol></li><li> T-75フラスコ1 mlトリプシン-EDTAを加えることによって細胞をトリプシン処理。 </li><li>トリプシン処理が完了した後、DMEM中の10ミリリットルの最終体積に細胞を再懸濁することにより、10％のFBS、2mMのL-グルタミン、1mMのピルビン酸ナトリウム、100 U / mlペニシリン及び100μg/ mlストレプトマイシンをトリプシンを中和する。 </li><li> 100μmのセルストレーナーで濾過して細胞が塊を除去した。 </li><li> DMEM中7.5×10 6細胞/ ml、10％FBS、2mMのL-グルタミン、1mMピルビン酸ナトリウム、100 U / mlペニシリン及び100μg/ mlストレプトマイシンの最終濃度で細胞を再懸濁細胞を数える。 </li></ol> 5。管形成アッセイ<ol><li> BMEはプレムしないようにプレートをロードする前に、24ウェルプレート、冷蔵庫内または20〜30分間、氷上で1ミリリットルのヒントを配置aturely固化する。 </li><li>ピペットで24ウェルプレートの各ウェルに減少成長因子BMEを250μl。小出ししながら、最終的な停止にピペットを押下しないことにより、泡を作成しないようにしてください。十分なBMEがうまくの中心メニスカスの形のまま​​であるように注意してください。マトリックスが薄すぎると、細胞は単層を形成する。 </li><li> BMEを固めるために30分間37℃で24ウェルプレートをインキュベートし、5％CO 2。 </li><li> BMEが設定されると、再懸濁3B-11細胞（約75,000細胞）10μlの馴化培地の約300μlのミックス。 <ol><li>馴化培地および使用内皮細胞型に応じて播種した細胞の数のボリュームを調整し、活性を証明するために馴化培地の連続希釈液をテストする。比較セルが異なる条件下で増殖させた場合は、メディアを調整するために使用された細胞の数に馴化培地容積を正規化。 </li></ol></li><li>チューブウィルlは2-4時間以内に形成され始める。馴化培地中の血管新生因子の濃度に依存して、ピーク管形成は、3〜12時間の間に発生し、アッセイのタイミングを最適化する必要がある。代わりに、不死化細胞の一次内皮細胞を扱う場合は、初期管形成は、数時間遅れで表示してもよい。 <ol><li>チューブは、多くの場合、18時間以内に悪化し始め、内皮細胞はアポトーシスを受ける。 </li></ol></li><li>ピーク管形成の際には、ウェルから慎重に吸引メディアはBME上の管ネットワークの中断を避けるため。 <ol><li> 1ミリリットルのDPBS、その後吸引して、各ウェルを洗浄する。 <ol><li>即時の可視化のために、細胞は、4.1.1のように、カルセインAMで染色した場合には、デジタルカメラ、または蛍光/共焦点顕微鏡に取り付けられた倒立顕微鏡を用いて各ウェルの画像管網に1ミリリットル新鮮なDPBSを追加します。 </li><li>後で視覚化のためのチューブネットワークを修正し、それぞれに4％パラホルムアルデヒドを追加するにはウェルを吸引し、室温で15分間インキュベートする。 <ol><li>パラホルムアルデヒドを取り外し、DPBSで二回洗浄し、各ウェルに1mLの新鮮なDPBSを追加します。 </li><li>前少数の管のような固定を顕微鏡イメージは、この手順の間に破損する可能性があります。 </li></ol></li></ol></li></ol></li></ol>チューブネットワークの6。定量<ol><li>調査者の好みに応じていくつかの異なる方法でチューブネットワークを定量化し、いくつかのコンピュータプログラムは、以下のパラメータを測定する能力を有する。管の数;ループ/メッシュ数;ブランチサイト/ノード数;チューブの長さ。 </li><li>そのようなチューブネットワークの定量化のための血管新生アナライザーのプラグイン16と画像jとして代表的なコンピュータプログラムを使用してください。 </li></ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146, 873-887 (2011).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438, 937-945 (2005).</li><li>Bouis, D., Kusumanto, Y., Meijer, C., Mulder, N. 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著者らは、開示することは何もない。

脈管形成は生理学的および病理学的プロセスの両方に関与している。血管新生に関与するメカニズムを研究することは、血管新生における主要な工程を再現アッセイの使用を必要とします。内皮細胞のチューブ形成アッセイは、他のアッセイに比べていくつかの利点を提供する。これは、アップを設定することが容易で比較的安価であり、時間内の細管を生成し、かつ定量化可能である。ま?...

血管新生、既存血管からの新しい血管の発達は、1〜3治癒 、器官の成長、胚発生、創傷を含めた多様なプロセスに不可欠である。新組織に内皮細胞、供給酸素と栄養が並ん血管を開発し、造血細胞による免疫監視を促進し、老廃物2,4を削除します。血管新生は、胚および胎児の発生の間に極めて重要である。しかしながら、この方法は、創傷治癒、骨格の成 ​​長、妊娠や月経周期1-3時の回の中を除いて、成人に休眠残る。 最後の20年間、血管新生を制御する重要な分子機構が出現し始めている。血管新生は、インテグリン、ケモカイン、アンジオポエチン、酸素検知剤、接合部の分子と内因性阻害5を含むプロと抗血管新生シグナルでバランスが厳密に調節イベントです。 proangいったんそのような塩基性線維芽細胞増殖因子（bFGF）、血管内皮増殖因子（VEGF）、血小板由来増殖因子（PDGF）、および上皮成長因子（EGF）などのイオ起源の信号は、内皮細胞は、内皮細胞が基底膜を分解するプロテアーゼを放出するレセプター活性化する。内皮細胞はその後増殖し、遊走、6,7日あたり数ミリメートルの速度で芽を形成する。 血管新生は、癌、乾癬、糖尿病性網膜症、関節炎、喘息、自己免疫疾患、感染症、およびアテローム性動脈硬化症8-10を含むさまざまな病的状態と関連している。による各種疾患における血管新生の重要性は、このプロセスは、より良い治療法の設計に重要である調節遺伝子および経路を理解する。 管形成アッセイは、血管形成に関与する遺伝子または経路を決定するための迅速かつ定量的な方法である。まず1988年に記載され、このアッセイの基礎をなす原則は、内皮細胞が分裂し、血管新生シグナルに応答11-13に急速に移行する能力を保持するということである。さらに、内皮細胞は分化し、基底膜抽出物（BME）のマトリックス上で培養管様構造を形成するように誘導される。これらの管は、接合部複合体により連結内皮細胞に囲まれた内腔を含んでいる。管形成はほとんどのチューブ量及び血管新生の刺激の種類に応じて2〜6時間以内に、このアッセイにおいて形成で迅速に起こる。 内皮細胞のいくつかのタイプの初代細胞および不死化細胞株14,15の両方を含むこのアッセイのために使用することができる。この記事に使用される細胞株は、マウス3B-11であったが、同じ方法論は、HUVEC（ヒト）細胞のようなSVEC4-10（マウス）のような他の内皮細胞株または初代内皮細胞と適用することができる。細胞株が使用されるかに依存し、内皮CEのかどうかLLSは、形質転換または非転換され、最適化は、適切な管形成に必要な理想的な時間を同定するために実施される必要がある。

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:339px;">Item</td>
			<td rowspan="2" style="width:189px;">Manufacturer</td>
			<td rowspan="2" style="width:124px;">Catalog #</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="93">
			<td height="114" rowspan="2" style="height:114px;width:339px;">Costar 24-Well Tissue Culture-Treated Plate</td>
			<td rowspan="2" style="width:189px;">Corning</td>
			<td rowspan="2" style="width:124px;">3524</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="169">
			<td height="190" rowspan="2" style="height:190px;width:339px;">BD Matrigel Basement membrane matrix, Growth factor reduced</td>
			<td rowspan="2" style="width:189px;">BD Biosciences</td>
			<td rowspan="2" style="width:124px;">354230</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="55">
			<td height="76" rowspan="2" style="height:76px;width:339px;">Gibco 0.05% Trypsin-EDTA</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25300-054</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="74">
			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25030-081</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">15140-122</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DMEM (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">11965-092</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DPBS (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">14190-144</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Fetal Bovine Serum</td>
			<td rowspan="2" style="width:189px;">Atlas Biologicals</td>
			<td rowspan="2" style="width:124px;">F-0500-A</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Calcein AM</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:124px;">C3100MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMSO</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
		</tr>
	</tbody>
</table>

endothelial cell tube formation assay for the in vitro study of angiogenesis

血管新生は、正常な組織発達および創傷治癒に不可欠なプロセスであるが、病理学的状態の多様な関連付けられている。このプロトコルを使用して、血管形成は、高速、定量化可能な方法でインビトロで測定することができる。一次または不死化内皮細胞馴化培地と混合し、基底膜マトリックス上に播種される。内皮細胞は、馴化培地中に見出さ血管新生信号に応答して、毛細血管様構造を形成する。管形成は、内皮細胞は、1時間とルーメンを含む細管を2時間以内に出始め以内に自分自身を整列し始めてすぐに発生します。チューブは、位相差倒立顕微鏡を用いて可視化することができ、または細胞が、蛍光または共焦点顕微鏡を介して可視化アッセイチューブに先立っカルセインAMで処理することができる。ブランチサイト/ノードの数は、/メッシュをループし、または形成された管の数または長さを容易にvitrでの測定値として定量化することができるO血管新生。要約すると、このアッセイは、迅速な再現可能な、そして定量的に血管形成の促進または阻害に関与する遺伝子および経路を同定することができる。

マウス3B-11内皮細胞は、固化した成長因子低減BME上に播種した - このアッセイにおいて、生成物マトリゲルを使用した - そして経時的に追跡。 図1に示すように、いずれのマウスのケラチノサイトまたは線維芽細胞によって分泌される血管新生因子は、経時管形成を誘導することができる。内皮細胞は、遊走し、メッキの1〜2時間以内に、小さな枝を形成し始める。最大管形成は?...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

管形成アッセイは、 インビトロ血管新生を測定するための高速、定量可能な方法である。内皮細胞は、馴化培地と混合し、基底膜抽出物でメッキされる。管形成は容易に定量時間と新たに形成された細管内で発生する。

のための内皮細胞チューブ形成アッセイ<em&gt;体外</em&gt;血管新生に関する研究

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

endothelial-cell-tube-formation-assay-for-vitro-study

Research

JoVE Journal

Biology

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

59.0K ビュー.  American University. 管形成アッセイは、 インビトロ血管新生を測定するための高速、定量可能な方法である。内皮細胞は、馴化培地と混合し、基底膜抽出物でメッキされる。管形成は容易に定量時間と新たに形成された細管内で発生する。 

ビデオ: Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

クリストファーヒューズ：血管新生の研究のためのin vitroモデル（インタビュー）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

血管新生の研究のために最適化されたフィブリンゲルビーズアッセイ

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

腫瘍細胞のVasculogenic活性を評価するためにマトリゲルベースの​​チューブ形成アッセイ

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

凍結保存サンプルの回復のための日曜大工のデバイスが誤って低温貯蔵タンクに落とし

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

ラット腸間膜具象化：血管新生に関与する細胞のダイナミクスを調べるためのモデル

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

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 ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

血管内皮増殖因子の評価のための簡易バイオアッセイ

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.