Name: development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production
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この作品は、国立心肺血液研究所によってサポートされていましたHL56237、糖尿病および消化器の国立研究所腎臓病研究所DK97391-03、国立科学財団（NSF-1227359およびEPS-1003907）を付与します。

1.マイクロ流体デバイスの製造<ol><li> SU-8 50マスター型の標準的なフォトリソグラフィ製作<ol><li>スピンコーティングする前に、シリコンウエハを清掃してください。 15分間イソプロピルアルコール（IPA）、続いて15分間アセトンで裸の2インチのシリコンウエハをすすぎます。 1時間150℃のホットプレート上に配置することによって、ウエハを脱水。脱水後、室温でウエハを冷却します。 </li>...

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<html>この記事では、の[Ca 2+] iおよびNO産生が、マイクロ流体デバイスを用いて培養微小血管ネットワーク、ECジャンクションおよび細胞骨格F-アクチン分布の特徴付け、およびECの定量的測定の開発のための詳細なプロトコルを提示します。灌流マイクロ流体デバイスは、生体内微小血管の形状とせん断流動条件の近いシミュレーションを可能にするin vitroモデル?...

インビボでの血管壁の内側を覆う内皮細胞（EC）は、常に流れるように露出しているが、培養された電気部品は、多くの場合、静的な条件下で増殖させ、炎症促進性の表現型1,2を示しています。マイクロフルイディクス技術は、所望の流れ条件の下で成長し、特に血管のECのために、培養細胞のための機会を提供する幾何学的に拘束されたマイクロスケール（サブミリメートル）チャネル3を介して正確に制御された流体を可能にします。これらの機能は、従来の静的な2D細胞培養物よりもin vivoでの細胞培養条件近づけます。彼らは、マイクロ流体デバイスは、血管系の種類をモデル化するために使用され、機械的および/または化学的な刺激に対するEC応答を研究する際に非常に重要です。 静的な細胞培養、生物医学Fにおける適応とマイクロフルイディクスのアプリケーションの上にマイクロチャネルネットワークによって実証利点にもかかわらずieldは限られたままです。最近のレビューによって報告された、この分野（85％）の出版物の大半は、エンジニアリングジャーナル4に残っています。マイクロ流体デバイスの性能は、ほとんどの生物学者は、このようなトランスウェルアッセイおよびこの小型化されたデバイスへのマクロスケール培養皿/ガラススライドのような現在の技術からスイッチするための十分な説得力はなかったです。マイクロフルイディクスは、前方にこのフィールドを移動するには学際的なコラボレーションを必要とする学際的なフィールドです。この技術的な記事の目的は、生物学的なアプリケーションおよびマイクロ流体微小血管の機能的検証を提供しながら、専門分野間の知識のギャップを減らし、生物学者によって製造手順が理解できるようにすることです。可視化実験プロトコルは、マイクロ流体デバイスとエンジニアと生物学者の間で緊密な連携を表し、それらの生物学的ユーティリティ、両方の製造を含みます。 我々は最近、いくつかの報告しましたマイクロ流体素子5でのin vitro微小血管ネットワークを使用して生物学的用途。適切マイクロチャネルネットワークの寸法を設計し、所望のせん断応力を適用するためには、数値モデルは密接に流れプロファイルを推定する計算流体力学ソフトウェアで構築されました。 すなわちコンフルエンスに達したマイクロチャンネル中に播種した一次ヒト臍帯静脈内皮細胞（HUVEC）を、連続灌流で3~4日で、マイクロチャネルの全内面を覆っ。適切なバリアの形成はVE-カドヘリン染色によって実証および静的細胞培養条件下で無傷の微小血管内に形成されたものと比較しました。個別に灌流し、無傷の微小血管6-8で開発された実験プロトコルを適用することにより、我々は定量的に蛍光とアデノシン三リン酸（ATP）に応答して、ECの変化の[Ca 2+] iおよび一酸化窒素（NO）の産生を測定しましたdicators共焦点と従来の蛍光顕微鏡。 ECにおけるアゴニスト誘導増加は、の[Ca 2+] iおよびNO産生は、微小血管の透過性6-15における炎症性メディエータによって誘発される増加のために必要な細胞内シグナルとして報告されています。いくつかの以前の研究は、DAF-2 DAロードマイクロ流体デバイス16,17の画像を示したが、適切な解像度およびデータ分析はまだ18を達成していませんでした。我々の知る限りでは、この研究は、内皮におけるアゴニスト誘導性の動的変化の[Ca 2+] iとNO産生利用マイクロ流体ベースのシステムの最初の定量的測定を示しています。 微細加工技術は、ダウンして数ミクロンのマイクロチャネルを作製し、in vivoでの微小血管系の幾何学的構造を模倣するために複雑なパターンの開発を可能にする柔軟性を持っています。ここでは、分岐、3つのレベルを持つ典型的なマイクロチャネルネットワークを提示しました。このネットワークは、微細加工クリーンルーム及びソフトリソグラフィーで行われるフォトリソグラフィ技術の組合せによって製造されます。

<table border="0" cellpadding="0" cellspacing="0" width="1058">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:303px;">ATP</td>
			<td style="width:247px;">Sigma-Aldrich</td>
			<td style="width:327px;">A2383</td>
			<td style="width:183px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetone</td>
			<td>Fisher Scientific</td>
			<td>A929</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biopsy punch</td>
			<td>Miltex</td>
			<td>33-31 AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Albumin</td>
			<td>MP Biomedicals</td>
			<td>810014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Brain Extract (BBE)</td>
			<td>Lonza</td>
			<td>CC-4098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover-slip</td>
			<td>Fisher Scientific</td>
			<td>12-542C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAF-2 DA</td>
			<td>Calbiochem</td>
			<td>251505</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dextran</td>
			<td>Sigma-Aldrich</td>
			<td>31390</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-Goat IgG (H+L) Secondary Antibody</td>
			<td>Life technologies</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DRAQ5 (nuclei staining)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>4084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Endothelial Cell Growth Supplement (ECGS)</td>
			<td>Sigma-Aldrich</td>
			<td>E2759-15MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Gibco</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluo-4 AM</td>
			<td>Life technologies</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin from porcine skin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin (50 mg/mL)</td>
			<td>Gibco</td>
			<td>15750-078</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-548B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Pasteur pippette</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin sodium salt from porcine intestinal mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-10KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES Buffered Saline Solution</td>
			<td>Lonza</td>
			<td>CC-5024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human umbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>CC-2517</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropyl alcohol (IPA)</td>
			<td>VWR</td>
			<td>89125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mammalian Ringer Solution Ingredient</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl (132 mM)</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCL (4.6 mM)</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2 &#183; 2H2O (2.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4 &#183;7H2O (1.2 mM)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose(5.5 mM)</td>
			<td>Fisher Scientific</td>
			<td>BP350-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">NaHCO3 (5.0 mM)</td>
			<td>Fisher Scientific</td>
			<td>S233-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Salt (9.1 mM)</td>
			<td>Research Organics</td>
			<td>6007H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes Acid (10.9 mM)</td>
			<td>Research Organics</td>
			<td>6003H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MCDB 131 Culture Medium</td>
			<td>Life technologies</td>
			<td>10372-019</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin (F-actin staining)</td>
			<td>Sigma-Aldrich</td>
			<td>P1951</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline&#160;</td>
			<td>Life technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scalpel</td>
			<td>Exel Int</td>
			<td>29552</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scotch tape</td>
			<td>3M</td>
			<td>34-8711-3070-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silicon wafer</td>
			<td>VWR</td>
			<td>14672</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 photoresist</td>
			<td>MicroChem</td>
			<td>SU-8 2050 Y111072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tissue culture flasks</td>
			<td>Sigma-Aldrich</td>
			<td>Z707503-100EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Chemical Book</td>
			<td>T6878</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin Neutralizer solution</td>
			<td>Gibco</td>
			<td>R-002-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin/EDTA Solution (TE)</td>
			<td>Gibco</td>
			<td>R-001-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tubing</td>
			<td>Cole-Parmer</td>
			<td>PTFE microbore tubing, 0.012&#34; ID x 0.030&#34; OD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VE-cadherin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-6458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Name of Equipment</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biosafety Laminar hood</td>
			<td>NuAire</td>
			<td>NU-425 Class II, Type A2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CCD camera</td>
			<td>Hamamatsu</td>
			<td>ORCA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art</td>
			<td>F42022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>Wenesco</td>
			<td>HP-1212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubator</td>
			<td>Forma Scientific</td>
			<td>3110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isotemp oven</td>
			<td>Barnstead</td>
			<td>3608-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithography bench</td>
			<td>Karl Suss</td>
			<td>MA6 Contact Lithography</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical microscope</td>
			<td>Nikon</td>
			<td>L200 ND &#38; Diaphod 300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Shutter for the CCD camera</td>
			<td>Sutter Instrument</td>
			<td>Lambda 10-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>PVA TePla/Harrick plasma</td>
			<td>M4L/PDC-32G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Brewer Science</td>
			<td>Cee 200X</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump system</td>
			<td>Harvard Apparatus</td>
			<td>703005</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Software</td>
			<td style="width:247px;">Company</td>
			<td style="width:327px;">Catalog Number</td>
			<td style="width:183px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CAD software</td>
			<td>Autodesk</td>
			<td>AutoCAD 2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFD simulation software</td>
			<td>COMSOL</td>
			<td>COMSOL Multiphysics 4.0.0.982</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Images acquire and analyse for NO production&#160;</td>
			<td>Universal Imaging</td>
			<td>Metafluor</td>
			<td></td>
		</tr>
	</tbody>
</table>

development and characterization of in vitro microvessel network and quantitative measurements of endothelial [ca2+]i and nitric oxide production

インビボでの血管壁の内側を覆う内皮細胞（EC）は、常に流れるように露出しているが、培養された電気部品は、多くの場合、静的な条件下で増殖させ、炎症促進性の表現型を示しています。マイクロ流体デバイスの開発は、二十年以上のエンジニアに受け入れてきたが、それらの生物学的用途が限定されたまま。より生理学的に関連する生物学的用途によって検証試験管内微小血管モデル内のフィールドを進め、in vivoでおよびin vitroの研究で間のギャップを埋めることが重要です。ここで、我々は長期的な灌流能力を備えたマイクロ流体デバイスを用いて培養微小血管網の開発のための詳細な手順を提示します。また、共焦点と従来の蛍光顕微鏡を用いてリアルタイムでECの[Ca 2+] iおよび一酸化窒素（NO）の生産におけるアゴニスト誘発変化の定量的測定のためのアプリケーションを示しています。形成された微小血管ネット連続灌流との仕事は、ECの間でよく発達した接合部を示しました。 VEカドヘリン分布を静的培養EC単層よりも無傷の微小血管で観察されたものと近かったです。 EC過渡増加するATP誘発性の[Ca 2+] iとNO産生を定量的培養微小血管の機能性を検証し、個々の細胞レベルで測定しました。このマイクロ流体デバイスは、電気部品が近いin vivoでの従来の静的な2D培養に比べ細胞培養環境を作るよく制御され、生理学的に関連する流れ、下に成長することができます。マイクロチャンネルネットワーク設計は、非常に汎用性があり、製造プロセスが簡単で、再現性があります。デバイスは、容易に高解像度の画像化を可能にする共焦点又は従来の顕微鏡システムに一体化することができます。培養された微小血管ネットワークが初代ヒトのECにより形成することができるので、最も重要なのは、このアプローチはどのように調査するために有用なツールとして機能します病理学的に患者サンプルから変更された血液成分は人間のECに影響を与え、臨床問題への洞察を提供します。それはまた、薬物スクリーニングのためのプラットフォームとして開発することができます。

このセクションでは、このプロトコルを使用して開発した培養微小血管網で得られた結果の一部を示しています。マイクロチャネルのパターンは、3レベルの分岐ネットワーク（ 図1A）です。この設計では、4幅100μm娘チャネルに再び2 126ミクロン幅チャネル、および支店に159μmの広い母のチャネルブランチ。 3次元数値シミュレーションは、このマイクロ?...

Watch this Scientific Journal Video about Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production at JoVE.com

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

の開発と特性評価<em&gt;インビトロ</em&gt;微小血管ネットワークおよび内皮の定量的測定の[Ca<sup&gt; 2+</sup&gt;]<sub&gt;私</sub&gt;と一酸化窒素の生産

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

The overall goal of this procedure is to develop a microfluidic device that allows endothelial cells to grow under physiologically relevant shear flow and to measure the agonist induced changes in their calcium and and nitric oxide production. This message can help advance the future of microvessel research, by validating the in vitro microvessel model and bridging the gaps between in vivo and the in vitro studies. The main advantages of this technique are the versatile design of the microchannels to mimic various vasculature and they improve cell culture environment that is closer to in vivo than the conditional static to that original cultures.Demonstrating the procedures will be Sulei and Xiang, the two from my laboratory. Before beginning, use standard photolithography to create a master mold and soft lithography to fabricate the PDMS microchannel devices as described in the text protocol. To prepare the microchannel devices, first cover the inlet and outlet with a thin piece of PDMS and place the device inside a Petri dish with a wet tissue.Then, wrap the dish with Parafilm and sterilize the device under a UV light for at least three hours. The next step is to apply the fibronectin. First, place a drop of 30 to 50 microliters of PBS at the inlet.Create a vacuum at the outlet to rinse the device. Next, place a drop of 100 micrograms per milliliter fibronectin at the inlet. Create a vacuum at the outlet to load the fibronectin solution.Next, cover the inlet and outlet. Wrap the dish with Parafilm again and incubate the device overnight at four degrees Celsius. The next day, manually rinse the device with PBS three times.Then, load it with 30 to 50 microliters of cell culture media and warm up the device in an incubator at 37 degrees Celsius for at least 15 minutes. Begin by mixing HUVECs into HUVEC culture media with eight percent Dextran at two to four million cells per milliliter. The Dextran is needed to increase the viscosity and thus improve the cell seeding.Next, put 10 to 20 microliters of cells over the inlet and introduce them using gravity or by using capillary action from a glass pasteur pipette at the outlet. Next, incubate the device for 15 to 20 minutes. Then, check the seeding status under a microscope.If necessary, load more cells to achieve a desired cell density. Once enough cells are loaded, gently rinse the device with normal, warm cell culture medium to remove the Dextran. Then, culture the device without any media perfusion for six hours.Next, set up the tubing connections for the long term perfusion. Tape the device to a Petri dish and then connect the inlet tubing to a syringe with a needle. Insert tubing into both the inlet and the outlet of the device.Connect the outlet tubing to a waste collector. Now, place the dish and waste container in an incubator. Connect the syringe with a long inlet tube to an external perfusion pump that passes through the incubator door.Then, set the perfusion rate according to the experimental design. With a well controlled perfusion, the culture in the microvessel network can be maintained up to two weeks. Therefore, it can be extended to longer term studies mimicking the cellular and the hemodynamic environment on the different physiological and the pathological condition.To begin the immuno-staining, such as VE-cadherin labeling, first fix the cells by perfusing with two percent paraformaldehyde for 30 minutes at four degrees Celsius. After a series of perfusions to block, permeabilize, and stain with antibodies, keep the device at four degrees Celsius until the cells are imaged. To image the calcium concentration in the cells, perfuse the device with 10 milligrams per milliliter of albumin in Ringer solution for 15 minutes at 37 degrees Celsius.Next, perfuse the device with five micromolar Fluo-4 AM for 40 minutes at 37 degrees Celsius. Then, wash out the lumen bound Fluo-4 AM with albumin Ringers for 15 minutes at 37 degrees Celsius. Next, start up the confocal system for intercellular calcium level imaging with Fluo-4 AM.Acquire images of the Fluo-4 loaded microvessels.Collect the baseline images for 10 minutes. Then, perfuse the device continuously with 10 micromolar ATP and record the changes of fluorescence intensity for 20 minutes. To image nitric oxide production in the cells, perfuse the device with albumin Ringers for 15 minutes at 37 degrees Celsius.Then, perfuse the cells with 5 micromolar DAF-2 DA for about 35 to 40 minutes at 37 degrees Celsius. Next, set up the fluorescence imaging system to measure ATP induced nitric oxide production. Record the baseline for 5 minutes, then perfuse the device with 10 micromolar ATP and collect the images for 30 minutes at one minute intervals.For the cell nitric oxide measurement, it is important to understand that the detach adapter for intensity profile represents a cumulative nitric oxide production with time and not a nitric oxide concentration. Manually select the region of interests from the collected images at the individual cell level. Each ROI covers the area of one individual's cell which can be indicated by the floresense outline.Quantify the ATP induced changes in endothelial calcium and nitric oxide. By calculating the changes in the floresense intensity of Fluo-4, less the tissue background. Quantify the nitrous oxide production rate by conducting the first differential conversion of the floresense intensity of DAF-2 over time.The microchannel pattern in the device has a three level branching network. A 3D numerical simulation was performed to estimate the shear stress distributions under the flow rate of 0.35 microliters per minute. Endothelial cells were seeding into the network as described.Then, F-actin and nuclei were labeled. Similarly, VE-cadherin was also stained. The results were similar to the VE-cadherin expression in an intact venule.Next, ATP induced increases in intracellular calcium and nitric oxide production were examined. Calcium levels were examined using Fluo-4 AM as described in the protocol section. Nitric oxide production was monitored using DAF-2 DA as described in the protocol section.The results were comparable to those derived with individually perfused intact venules. The advanced was easy and tightly controlled in biological conditions and the dynamic fluid environments, which would not have been possible with conventional microskill techniques. After watching this video, you should have a good understanding of how to fabricate the device, and perform calcium and nitric oxide measurements.After this technique paved the way for researchers, not only to investigate underlying agonist induced but also to open broader applications for bio medical research.

Research

JoVE Journal

Biology

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

一酸化窒素とスピントラップを使用して、細胞からの電子常磁性共鳴分光法によるスーパーオキシドアニオンラジカルの検出

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生物学

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

リジン脱メチル化酵素活性の定量測定のためのMassSQUIRMのアプリケーション

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Analysis of Oxidative Stress in Zebrafish Embryos

ゼブラフィッシュ胚における酸化ストレスの分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

精製された野生型および変異型CFTRタンパク質の機能的再構成とチャネル活性の測定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

細胞内カルシウムの二光子イメージング<sup&gt; 2+</sup&gt;取扱い及び内皮における一酸化窒素産生および単離されたラット大動脈の平滑筋細胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

Quantitation of Endothelial Cell Adhesiveness In Vitro

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 ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

化学発光法を用いて、生物学的材料に、一酸化窒素経路に亜硝酸塩と硝酸塩、代謝物を測定します

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

遺伝的にコードされた蛍光一酸化窒素の応用（•NO）シングル細胞におけるNO•信号のリアルタイムイメージングのためのプローブ、geNOps、

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

定量的イメージング技術を用いたヘテロ細胞集団の識別と特徴付け

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...