著者らは、寛大にH1のMSC、と博士はティムのハッカー、博​​士Gouqingソングとウィスコンシン心臓血管生理学の大学の先生ジルコッホを提供するための博士Peiman Hemmatiを（医学科、ウィスコンシン大学マディソン校）に感謝いたしますマウスの手術を行うための中核施設。この作品は、ブライアンフリーマンとNIH R21 HL089679に大学院研究フェローシップを通じて、全米科学財団によってサポートされていました。

1。ドナー細胞のトランスフェクション<ol><li> 収穫間葉系幹細胞（親切に先生Peiman Hemattiから寄贈されたH1胚性幹細胞由来のMSCは、、代わりに、 生体内で融合する仮説任意の種の任意のセルのタイプを利用することができる）70 - 1Xのトリプシンコンフルエント80％（Mediatech、 5分間マナッサスVAは）。α- MEM完全培地（抗生物質フリー、Invitrogen社、カールスバッドカリフォルニア州）18でトリプシンを不活性化する。 5分間300 × gで遠心する。 </li><li>注意深く上清を吸引してから再サスペンドペレットの1X PBS 1mLにと血球計算盤を用いて細胞を数える。 </li><li> 1.5 mLの遠心チューブに移す1.5 × 10 6細胞。 5分間300 × gで遠心する。 </li><li>注意深く上清を吸引。 Rバッファー（ネオントランスフェクションシステム、Invitrogen社）とp231 pCMVe - betaAc - STOP - lucを（Addgene、ケンブリッジ、MA）6μgの（濃度2μg/ 5.0 × 10 5細胞）を300μLのペレットを再懸濁します。 Eバッファの代わりに3 mLの（Invitr製造業者のプロトコルごとのエレクトロポレーションのドッキングポート（ネオントランスフェクションシステム、Invitrogen社）にogen）。 </li><li> 100μLネオンのピペットチップへの細胞プラスミド溶液を移し、20ミリ秒と1500ボルトの大きさのパルスの持続時間とエレクトロ。場所は、9.7 mLのα- MEM完全培地を含む15 mLコニカルチューブに細胞をエレクトロポ。 </li><li>を10mLの総量を得るために1.5を追加で2回とプールトランスフェクトした細胞を繰り返します。 10 mLのα- MEM完全培地を含むT175フラスコに10 mLの細胞懸濁液（1.5 × 10 6細胞）を追加します。エレクトロポレーション後の細胞の生存率はT175あたり約4.5 × 10 5生細胞を得るために、約30％です。 </li><li>トランスフェクション後にα- MEM完全培地24時間を変更します。 </li><li> 80％コンフルエント（〜2 - - 3日エレクトロポレーション後）収穫は、70細胞をトランスフェクション。血球計算盤を用いて細胞数を実行し、1.0 × 10 6の濃度で細胞を再懸濁α- MEM完全培地のcells/50μL。最小化時の細胞は注入前に細胞死を減らすため懸濁液中で過ごす。 </li></ol> 2。心筋内注入<ol><li>すべてのセルで構成的に発現するCreリコンビナーゼ（B6.C - TG（CMV - CRE）1Cgn / J、ジャクソン研究所、バーハーバー、するように設計トランスジェニックマウスで（フェニックスファーマシューティカル社、セントジョセフ、ミズーリ）イソフルランで麻酔を誘導するME）。 </li><li>バリカンまたは化学的脱毛剤を使用して胸部から髪を削除します。 </li><li> 150μLのストローク量で毎分130回の呼吸 - 120マウスの人工呼吸器で18ゲージのカテーテル（ベクトンディッキンソン＆カンパニー、フランクリンレイクスNJ）と場所で挿管する。 </li><li>これにより、開胸術を生産する第四肋間隙を介して横方向の切開を加えます。 </li><li>心臓の可視化、1 mLシリンジ（Temuroメディカル株式会社、サマセット、ニュージャージー州）を用いてトランスフェクトされた細胞懸濁液の2つの25μL注入し、28ゲージの針（Bectを作る上ディッキンソン＆カンパニー、フランクリンレイクスNJ）。心筋内注入を容易にし、臓器に過度の損傷を防ぐために、針の頭〜90度に曲げる。 </li><li>注射後、肋骨や筋肉の層を閉じるには、吸収性縫合糸（例えば、vicryl）を使用します。縫合の皮は4から0ナイロンや絹を使用してクローズ。 </li><li>マウスは麻酔と抜管から回復することができます。 </li><li> 対照群では、トランスフェクトされていない細胞とトランスフェクト細胞を（代わりに、Creをマウスが融合しがちではないトランスフェクト細胞を受信）受信野生型マウスの同じ濃度を受信するのCreマウスを培地のみ注射を受けたのCreマウスを含める必要があります。 </li></ol> 3。バイオフォトニックイメージング<ol><li>イメージングの前に5〜15分、腹腔内（IP）の15 mg / mLのD -ルシフェリン（キャリパーライフサイエンス、マサチューセッツ州ホプキントン）のマウスの体重1g当たり10μLを注入する。 </li><li>誘導のために4％でイソフルランを経由してマウスに麻酔を誘発するD 1 - メンテナンス2％。 </li><li>フェイスマスクが1の投与によるイメージングボックスで仰臥位の場所をマウス - メンテナンス麻酔用イソフルラン2％（Xenogenバイオフォトニックイメージングシステム、ホプキントン、MA）。いくつかのマウスを同時に撮像することができます。発光シグナルの簡単な比較のための実験用マウスで画像偽のコントロールマウス。 </li><li>リビングイメージのソフトウェアを（Xenogen）を使用して、（通常は60秒、結果を参照）、適切な露光時間を設定します。マウスに合わせて感度の変化を防ぐために、画像全体の面積の整合性を保つために、画像の領域を設定します。 4.5センチメートルの被写体の高さを設定します。 </li><li>ビューのフィールド内にマウスまたはマウスに対応する発光強度の信号を取得し、変更されていない画像ファイルを保存します。プロセスイメージは、コントロールまたはunmanipulatedマウスに対応したバックグラウンド信号を除去する。背景上の強度値は、動物内で融合細胞に​​対応しています。強度解析は、リビングの画像ソフト（Xenogen）またはオープンソースの画像解析ソフトウェアを用いて行うことができる。 Typically、関心領域は、（ROI）の動物と実験との間で強度のデータを比較するために選択されています。 </li></ol> 4。代表的な結果 Xenogenバイオフォトニックイメージングシステムの感度は、恒常的にC57/BL6マウス（ジャクソン研究所）の心筋に配信されました（231 - LUC - D3H1、Xenogen）ルシフェラーゼ発現する細胞株を決定する。 細胞を1 xの濃度で注入した10 6、1 × 10 3、または1 × 10 1セル。セルの製品納入後、6時間、マウスは、ルシフェリンを腹腔内注射し、Xenogenシステムを用いて画像化した。特定の信号は、1,000細胞（画像化された6マウスの2、 図2）で検出されたが、検出は10,000細胞（画像化された6匹の6）で、より信頼性が高くすることができます。重要なのは、この研究はまた、ルシフェラーゼ発現細胞と信号強度の数との間の大まかな相関関係を確立するのに役立った。 と Cre発現マウスの心筋に供給されるトランスフェクションした。細胞送達後の約1週間は、マウスが最初にD -ルシフェリンを注入することなく Xenogenシステムを用いて画像化した。予想通り、酵素基質なしで、ない強度の信号が（ 図3）は検出されなかった。次に、D -ルシフェリンを腹腔内投与し、ルシフェラーゼの強度に応じた信号を注入したため、細胞融合は、テストされた2つの4のマウスで検出された。同様の信号は、 このケースでは、研究は、心臓と周囲の組織の評価を可能にするために終了しました 。MSC -結合融合製品はin vivoで維持することができます示唆し、1週間後（ 図3）検出されたが、一方は想像ができる長期的な分析およびメンテナンス、増殖を追跡するために、より頻繁なイメージングマウスの融合製品の開発は、おそらく移行。 <img alt="図1" src="/files/ftp_upload/3581/3581fig1.jpg" /> 図1。 インビボでの細胞融合を検出する手法の概略図。floxedルシフェラーゼが発現するプラスミドをCreを発現するマウスの細胞と移植された細胞との融合が発生した場合は、ルシフェラーゼを発現されます。ルシフェラーゼは、マウス、次にイメージングマウスをXenogenバイオフォトニックイメージングシステムを用いて（19日から適応）に、酵素基質、D -ルシフェリンを注入することによって検出することができます<img alt="図2" src="/files/ftp_upload/3581/3581fig2.jpg" /> 図2。バイオフォトニックイメージングによる心臓組織におけるルシフェラーゼ発現細胞の検出の感度 。構成的に（231 - LUC - D3H1、Xenogen）ルシフェラーゼ発現する細胞株は様々な総細胞数でのC57/BL6マウスの心筋内のスペースに配信されました。マウスの代表的なイメージが再1 × 10 6、1 × 10 3と1 × 10 1細胞を（左から右へ）ceivingことが示され、画像は約6時間注入後に行った。 <img alt="図3" src="/files/ftp_upload/3581/3581fig3.jpg" /> 図3。細胞融合の暗示インビボ発光での定量化。MSCは、プラスミドつのloxP -ストップ- loxP部位-ルシフェラーゼおよび Cre発現マウスの心筋に供給されるトランスフェクションした。約1週間と細胞送達後2週間は、Creをマウスに細胞融合を示す発光の強度を測定するためにXenogenバイオフォトニックイメージングシステムを用いて画像化した。偽とマウス1-4の発光の写真と強度の（A）オーバーレイ（左から右へ）細胞送達後17日間。 （B）シャムとマウス1-4の発光の強度（左から右へ）細胞送達後17日間。関心領域は、注射部位と強度のLに相当する（黄色）に選ばれましたevelsはImageJの（フリーソース）ソフト20を使用して測定した。 （C）発光の強度は、全ての実験条件に対して偽マウスの利息の同じ領域に正規化された。一週間で、マウスの3と4は、マウスの細胞と移植MSCの自発的な融合を示唆して正の発光シグナルを示した。信号は2週間後にマウス3に持続した。マウス3に対応する信号の臓器特異的局在を決定するために、胸腔が露出し、主要な臓器が摘出し、画像化した。写真とマウス3の発光の強度の（D）オーバーレイ。小腸における強度信号の局在に注意してください。マウス3の発光の（E）強度。写真と偽マウスの発光の強度の（F）オーバーレイ。偽マウスの発光の（G）強度。

<ol>
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利害の衝突は宣言されません。

方法は、小動物を含めて、生物で、初めて、可能にするここで細胞融合の離散的な同定と時間的な分析を説明した。アプローチは、その後のバイオフォトニック画像解析でCre - loxP組換えを兼ね備えています。アプローチだけでなく、細胞 - 細胞融合だけでなく、ウイルスの細胞融合を追跡に適しているので、ウイルス感染を追跡するのに役立つ可能性があります。画像解析は急速であり、そ...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ネオントランスフェクションシステム</td><td>インビトロジェン、カールスバッド、カリフォルニア州</td><td> MPK5000 </td><td></td></tr><tr><td>ネオン100μLキット</td><td>インビトロジェン、カールスバッド、カリフォルニア州</td><td> MPK10025 </td><td> RとEバッファが含まれています</td></tr><tr><td> - MEM粉末</td><td>インビトロジェン、カールスバッド、カリフォルニア州</td><td> 12000-022 </td><td></td></tr><tr><td>ウシ胎児血清（FBS） </td><td> Hyclone、ローガンユタ州</td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C - TG（CMV - CRE）1Cgn / J </td><td>ジャクソン研究所、バーハーバー、ME </td><td> 006054 </td><td></td></tr><tr><td> 10Xトリプシン</td><td>フィッシャーサイエンティフィック、フォレストローン、ニュージャージー州</tD&gt; <td> MT - 25 - 054 - Clを</td><td></td></tr><tr><td> L -グルタミン</td><td>フィッシャーサイエンティフィック、フォレストローン、ニュージャージー州</td><td> 25030-081 </td><td></td></tr><tr><td> D -ルシフェリン</td><td>キャリパーライフサイエンス、マサチューセッツ州ホプキントン</td><td> 122796 </td><td></td></tr><tr><td> Xenogenバイオフォトニックイメージングシステム</td><td>キャリパーライフサイエンス、マサチューセッツ州ホプキントン</td><td> IVISスペクトラム</td><td></td></tr><tr><td>ナトリウムBiocarbonate </td><td>シグマアルドリッチ、セントルイス、MO </td><td> S6014 - 500G </td><td></td></tr><tr><td>非必須アミノ酸</td><td>インビトロジェン、カールスバッド、カリフォルニア州</td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

融合し、同じタイプの2つ以上のセルの能力は、骨格筋、骨、胎盤を含む多くの複雑な器官を形成し、進化の過程で後生動物に利用されている。現代の研究では、同じタイプの拡張機能を与えるの細胞の融合を示しています。例えば、胎盤のヒューズの栄養膜細胞が合胞体栄養細胞を形成する際に、合胞体栄養細胞はより未融合のトロホブラスト1月4日より母体胎児関門の栄養素やホルモンを輸送することができます。より最近の研究では、異なる種類の細胞の融合は、細胞の運命を導くことを実証。融合による&quot;復帰&quot;または細胞の運命の変更は、いったん細胞培養系に限定されると考えられていた。しかし、幹細胞移植の出現は、私たちが発見につながったと幹細胞を他の人は、in vivoでの体細胞と融合することができ、その融合は、幹細胞の分化5-7を容易にします。従って、細胞融合は、規制プロセスのcですしたがって、細胞の生存と分化を促進するのapableとさらに発展、組織の修復と疾患の病因の中心的な重要性の可能性があります。 適切な技術の不足が）1に、細胞融合の研究をされている制限することで、正確に時間をかけて）、トラックの融合製品を融合製品を識別し、2。ここでは、融合時に生物発光の誘導（ 図1）を経由して、両方の制限に対処するために新しいアプローチを提示する、生物発光は 、in vivo 8-15 の高感度で検出することができます私たちは、ホタルルシフェラーゼ（Photinusスピラリス ）遺伝子は隣接して配置符号化構造を利用する 。 loxP配列で挟まれた。細胞は、Creリコンビナーゼの蛋白質を発現する細胞でこの遺伝子のヒューズを表現するときは、 つのloxP部位が切断されると停止信号がルシフェラーゼの転写を可能に切除されているが、信号がinduciなので、終止コドンBLE、偽陽性シグナルの発生率は非常に低いです。のCre / loxPをシステム16、17を利用する既存の方法と異なり、我々は&quot;生きて&quot;検出信号を取り込んでいるし、それによって初めて、生体内で細胞融合の動態を追跡する機会を与える。 アプローチを実証するために、普遍的にCreリコンビナーゼを発現するマウスは、floxedストップコドンのルシフェラーゼ下流を表現するための構築物でトランスフェ幹細胞の受信者を務めた。幹細胞は、心筋内注射により移植し、移植生体内の画像解析は、の存在を追跡するために実施された後に時間をかけて心と周囲の組織の融合製品。このアプローチは、開発、疾患や成人の組織修復の任意の段階で任意の組織型における細胞融合を分析するために適合させることができる。

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

時間の経過とともに生物の細胞融合を追跡する方法について説明する。アプローチは、Creを利用し<em&gt;つのloxP</em&gt;再結合は、細胞融合によりルシフェラーゼ発現を誘導する。生成された発光シグナルは、末梢組織で〜1,000細胞の検出の感度バイオフォトニックイメージングシステムを用いて生体内で検出することができます。

細胞融合の検出のためのCre - LOX Pの組換えのアプローチインビボでの</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K ビュー.  University of Wisconsin-Madison. 時間の経過とともに生物の細胞融合を追跡する方法について説明する。アプローチは、Creを利用しつのloxP再結合は、細胞融合によりルシフェラーゼ発現を誘導する。生成された発光シグナルは、末梢組織で〜1,000細胞の検出の感度バイオフォトニックイメージングシステムを用いて生体内で検出することができます。

ビデオ: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

干渉反射イメージングセンサー（IRIS）を用いた生体分子の検出

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

生物工学

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. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

のためのシンプルなマイクロ流体デバイス<em生体内で&gt;</emの&gt;イメージング<em&gt; Cエレガンス</em&gt;<em&gt;ショウジョウバエ</em&gt;とゼブラフィッシュ

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

マイクロインジェクションアッセイをキズや<em&gt;インビボ</em&gt;表皮の局在は、応答記者傷<em&gt;ショウジョウバエ</em&gt;胚。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

単一細胞分泌物の時空間イメージング用ラベルフリー技術

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

用中空糸バイオリアクター<em&gt;インビボ</em&gt;哺乳動物の組織培養様

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D相互作用の研究のための3Dコラーゲンゲルおよびマイクロチャネルの作製<em&gt;インビボ</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

キャピラリー配列で複数の核酸の視覚的検出

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...