Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

TiO 2の調製は、ガラスカバースリップをコーティングを採用しました<ol><li>ナンバーダイヤモンドスクライバーを使用してカバースリップ。これは、各カバースリップを追跡するためだけでなく、サンプルの正しい側が上を向いていることを確実にするためだけではなく役立ちます。次にピラニア溶液にそれらを浸漬することにより、第一のddH 2 Oを実行して下に、カバーガラスを清掃し（H 2 SO 4：H 2 O 2 = 4：1）。 10分後、のddH 2 Oに、徹底的に8回をカバースリップをすすぎN 2気流下でカバーガラスを乾燥させます。 </li><li>無線周波数（RF）スパッタリング装置にTiO 2のターゲットを設定します。ポリイミドテープを用いたスパッタリング装置の試料ホルダー上にカバーガラスを取り付けます。スパッタチャンバー内の試料ホルダーを配置します。圧力が2.0×10 -4 Paのに達するまで室を避難させます。 </li><li>チャンバー内にArガスを導入し、4.0トルの堆積圧力を設定します。シャッターが閉じ維持しながら、徐々に増加W. 70へのRF電力</li><li>シャッターを開き、120〜150ナノメートル（ 図1：ステップA）の厚さのフィルムを得るために15分間スパッタ。膜の成長速度は、マシンごとに導出する必要があります。 </li></ol>セル・撥水膜との2表面コーティング<ol><li>プラズマ反応器のメーカーが提供する説明書に従って、O 2プラズマで試料を処理することにより、TiO 2の表面を親水化。私たちは、100 SCCMのO 2流量で200 Wで5分間サンプルを処理します。 ddH 2 Oにサンプルを浸し、表面が超親水であることを確認します。 N 2気流下で完全に表面を乾燥させます。 </li><li> 100ミリリットルのトルエンに39.6μlのOTSを追加することによって、1mMのオクタデシルトリクロロシラン（OTS）溶液を調製します。室温で1時間、溶液中の試料を浸し。 N 2 -filledグローブバッグ（：ステップB 図1）の内部では、この手順を行ってください。 </li><li>物理吸着分子を除去するために、sonicat電子トルエン、アセトン、エタノール中のサンプル、及びその順序で各5分間、のためのddH 2 Oを。新鮮なのddH 2 O中でサンプルを4回すすぎ、N 2気流下で表面を乾燥。表面は100〜110°の接触角を有する疎水性でなければなりません。 </li></ol> 3. 現場外表面パターニング<ol><li>層流フードでの作業、表面にダイヤモンドスクライバーで数スクラッチマークを描きます。マークが処理された領域を追跡し、また焦点に顕微鏡をもたらすのに役立ちます。 5分間70％エタノール中にサンプルを浸漬することにより被覆されたOTSのTiO 2を殺菌します 。その後、滅菌のddH 2 Oで二回サンプルをすすぎ</li><li> 35-mmディッシュ内のサンプルを置き、PC12成長培地2mlを追加し（ステップ4.2を参照）。 CO 2インキュベーター（37℃）で3時間以上インキュベートします。この手順は、血清アルブミンの表面上に吸収させることを意図しています。吸着アルブミンは他のproteのその後の吸着を阻害しますインと細胞（ 図1：ステップC）。 </li><li>待っている間に、倒立蛍光顕微鏡を設定します。 <ol><li> 、アークランプをオンにするUVフィルターキューブを挿入し、20X対物レンズを設定します。 </li><li>光強度Iを測定する（W cm -2の）紫外線強度計を用いて、 および dの用量のために（秒）照射時間Tを算出する（Jセンチ-2）として：T = D / I。 例えば、200 J cmの線量で照射すること-2 600ミリワットのcm -2での光源を用い、照射の333秒が必要です。 </li><li>ステージマイクロメータを使用して、 例えば 、照射される領域のサイズを設定するには、200ミクロンの視野絞りを閉じます。 </li></ol></li><li> 3時間のインキュベーションの後、3.0 mgのmlの200μlの-1 IV型コラーゲン（;最終濃度は300μgmlの-1 COL-IV）を含む培地を補います。 </li><li>顕微鏡ステージに35-mmディッシュを転送します。スクラッチメートルを探しますアークは、試料表面上に顕微鏡の焦点を合わせると、200 Jセンチ-2（ 図1：ステップD）の投与量でUV光を照射します。 UV照射の領域は、視野絞りの開度を調整することにより、または仲裁ジオメトリのメタルマスクを用いて視野絞りの交換のいずれかによって変更することができます。 </li><li> （COL-IVなし）新鮮な増殖培地で培地を交換し、バックインキュベーター内のサンプルを配置します。 </li></ol> 4.細胞培養<ol><li> PC12細胞のルーチン培養物を、コラーゲンコートされたプラスチックディッシュ上で行われます。 <ol><li>コートCOL-IVと60 mmの組織培養皿に、最初のddH 2 Oで表面を濡らし、すべてのddH 2 Oを吸引</li><li>元の溶液を希釈して300μgのmlの-1 COL-IVを準備するのddH 2 Oで（3ミリグラムml -1の）10倍</li><li> 300μgのmlの200μlの-1 60-mmディッシュあたりCOL-IVを追加します。表面を介して広がるソリューションをしてみましょう。 </li><li>ドライトン彼は約1時間、層流フード内で皿。 </li><li>ダルベッコのリン酸緩衝生理食塩水（D-PBS）4mlで二回表面をすすぎます。すぐにコートディッシュを使用しない場合、のddH 2 Oの4ミリリットルで一回の皿をすすぎddH 2 Oを吸引した後、インキュベーターで料理を格納します。それが2週間以上保存され維持しないようにしてください。 </li></ol></li><li>ダルベッコ改変イーグル培地（低グルコース）+ 10％ウシ胎児血清+ 5％ウマ血清+ 1％ペニシリン - ストレプトマイシン溶液：PC12細胞から成る増殖培地中で増殖させます。 CO 2インキュベーター中で細胞をインキュベートする（37℃、5％CO 2）、一日おきに培地の半分を交換します。継代細胞は、それらがコンフルエンスに到達する前に。 <ol><li>吸引は、増殖培地、及び追加の2 mlのPBS +（D-PBS +の10mg ml -1の牛血清アルブミン（BSA）+ 10mMのEDTA）を37℃に予熱。 37℃で5分間インキュベートします。静かに、すべてのCEを切り離すために料理をタップLLS。 </li><li> 15ミリリットルコニカルチューブ内の細胞を収集します。 37℃に予熱し、新鮮なD-PBS 3mlで料理を、洗浄します。 </li><li> 4分間150×gでチューブを遠心。 </li><li>上清を吸引除去し、成長培地1mlを加えます。 </li><li> 1：3：ルーチン培養のため、細胞を1分割5。 </li></ol></li><li> UV-修正OTS / TiO 2のサンプルの細胞をプレートに、細胞密度をカウントし、35-mmディッシュで3.0×10 5細胞 （ 図1：ステップE）を追加します。 1~2日間の加湿インキュベーター（37℃、5％CO 2）で皿をインキュベートします。両方のナイーブおよび神経成長因子（NGF）-differentiated PC12細胞は、パターニング実験に使用することができます。 NGF分化のために、7S-NGFの100 ngのmlの-1は数日間試料上に細胞をプレーティングする前に増殖培地に添加されます。 NGF分化は、PC12細胞の密着性を高めるように見える、特にその場でパターニング e。で、扱いやすくなりますxperiments。 </li></ol> 5. その場表面パターニング<ol><li> 現場外改変された表面上での培養の1〜2日後、細胞を取り付け、紫外線照射された領域のみに成長していることを確認します。ステップ3.4で説明したように、顕微鏡を設定します。 </li><li>成長培地を含む新しい35-mm組織培養皿にサンプルを転送し、100 ngのmlの-1 NGF（NGF分化細胞を用いた場合）、および100μgmlの-1 COL-IV。 </li><li>顕微鏡ステージ上の皿を置きます。適切な位置を見つけ、200のJ cm -2の（ 図1：工程F、G）の線量のUV光を照射します。新しく作成された許容領域は、 図1のアスタリスクで示されます。ステップG. </li><li> 30分以内に単一のサンプルの処理を完了してみてください。照射後、COL-IVなしにバック中にサンプルを転送します。 </li><li> Cuと料理を運ぶ時は十分に注意してくださいltured細胞や皿からサンプルを転送するには、パターン化された細胞の着脱防止するために皿に。 </li></ol><img alt="図1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> 全体的なプロセスの1模式図を図。詳細は本文を参照してください。 <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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The authors have nothing to disclose.

我々の現在のプロトコルでは、TiO 2の膜は、RFマグネトロンスパッタ法により形成しました。それは、私たちは再現性のサブnmの粗さと光触媒の TiO 2膜を作製することができますので、我々は、堆積のこの方法を好みます。スパッタ成膜プロセスは材料科学者や電子技術者によく知られているが、それは生物学者に非常にアクセスできないことがあります。その場合には、スピ...

半導体リソグラフィプロセスおよびその誘導体- 、フォトリソグラフィ1,2、電子ビームリソグラフィ3-6、および7-10を印刷マイクロコンタクトとしては-今定義された位置と形状に生きた細胞を成長させるために、細胞生物学の確立ツールとなっています。パターニング方法は、非許容バックグラウンドでセル許容コーティングのミクロの島からなる、微細加工された基板の使用に依存しています。このような基板は、パターンセルをするためのテンプレートとして機能します。これらの技術は、私たちの細胞の本質的な特性を抽出すること、及び細胞ベースの薬物スクリーニング11のスループットを高めるために、単一および多細胞レベルでの細胞およびそれらの機能を設計するための新規な方法を提供しています。 テンプレートのパターン形状を、すなわち 、 その場で変更することができれば、細胞がS上で培養されながら、自由度のセルのパターニングでは大きく増加しますurface。これらは大気中または真空中でサンプルを処理するためのパターン形成のための従来の方法は、直接、ここでは適用できません。そのため、様々な新しい表面改質技術はちょうど少数を示すために、光反応性化合物12,13またはレーザーアブレーション5,14に、 例えば 、基になっている、提案されています。提案された方法はうまくチョイら 16と中西17によって、より最近になって。Robertusらによって 15を見直し、されています。 ここでは、この記事では、我々は、二酸化チタン上に有機分子の光触媒分解を利用していますin-situで表面修飾、（TiO 2の）表面18,19の新規のプロトコルを記述します。この方法では、TiO 2のフィルムは、ガラス基板と細胞膜の間に有機インターフェースに挿入され、有機膜が局部的に紫外線（UV）を照射することにより、その場で分解され関心領域（λ&lt;388 nm）での光。新たなプロトコルは、細胞外マトリックスタンパク質および生細胞の両方の現場外のその場での微細パターンを作成するために使用できることを示します。 TiO 2は、細胞培養実験に導入することがフレンドリーになる特徴、そのうちの、生体適合性の化学的に安定し、光学的に透明です。このプロトコルは、細胞培養環境での細胞培養足場を修正するための材料科学ベースの代替手段を提供します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

二酸化チタン（TiO 2）の表面に吸着した有機汚染物質は、紫外線（UV）光の下で光触媒により分解することができます。ここでは、局所的に、基板表面の細胞親和性を変化させるためにTiO 2の光触媒を採用した新たなプロトコルを記述します。この実験では、薄膜のTiO 2膜をスパッタコーティングしたカバーガラス上で、TiO 2の表面は、続いて、細胞接着を阻害オクタデシルトリクロロシラン（OTS）に由来するオルガノシラン単層で修飾しました。サンプルは、細胞培養培地中に浸漬し、UV光を八角形の領域に照射された焦点を合わせました。神経細胞株PC12細胞を試料上にプレーティングしたときに、細胞は、UV照射領域に接着しました。我々はさらに、細胞を基板上に成長している場合でも、この表面改質はまた、 例えば 、 その場で行うことができることを示しています。表面の適切な修飾は、細胞外マトリックスpを必要とUV照射時に培地中に存在することが、コラーゲンをrotein。ここに提示された技術は、潜在的に共培養システムを構築するか、任意の培養で細胞を操作するための複数の細胞型のパターン化に用いることができます。

図2Aは、スパッタ堆積のTiO 2膜の断面の走査型電子顕微鏡（SEM）像を示します。観察から、フィルムの厚さは150nm程度であると推定されました。ここで顕著な堆積のTiO 2膜の平坦性があります。原子間力顕微鏡（AFM）によるさらなる分析は、表面の二乗平均平方根（RMS）粗さは0.2nmで（ 図2B）であることが明らかになりました。 <su...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

TiOを使用して水性環境中のタンパク質や細胞を光パターニング<sub&gt; 2</sub&gt;光触媒

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Research

JoVE Journal

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

7.8K ビュー.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

ビデオ: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

核酸、タンパク質と低分子のReagentless検出用電気化学DNAバイオセンサーの作製

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

生物工学

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

ネガティブステインEMによるタンパク質と高分子複合体の可視化：グリッドの準備から画像取り込みに

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

水性環境で炭化水素の変換を有効にするツールキット

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChipを使用して、ルート環境の急激な操作によるシロイヌナズナの根の成長のタイムラプス蛍光イメージング

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

水性二相系を用いた細胞の共培養パターニング

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

発光共鳴エネルギーは、哺乳動物細胞で発現膜タンパク質の立体構造変化を研究するために転送

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

マイクロリアクター及び、電磁作動に水滴が酵素として使用

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

パターニングタンパク質および細胞の多彩な方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

熱と圧力を用いた水性溶媒和による組み換えクモ絹の材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...