このプロジェクトは、MITの技術革新のためのデシュパンデセンター（RKとJMK）と化学と生物学的分離プログラムを通じてRKにNSFのキャリア賞0952493によってサポートされていました。我々は兵士のナノテクノロジー（ISN）とその施設の使用のためのMITのマイクロ技術研究所（MTL）のための研究所に感謝する。

1。 HL60細胞のローリング 1.1。加工基板の作製。 <ol><li>金でコーティングされたガラススライド上にPEG分子の自己組織化単分子膜を（SAM）が交互にするマイクロコンタクトプリンティング（μCP）4-7を使用する：α= 10の傾斜角度で受容体のパターンを定義したマイクロコンタクトプリンティングポリジメチルシロキサン（PDMS）スタンプの作製SU - 8の成形プロセスによる°。 20分間ピラニア溶液（30％過酸化水素に硫酸の3:1混合物）で、金表面を清掃し、使用する° Cより前の24.5で、大量のDI水で表面を洗う。エタノール5mmのPEG溶液とインクPDMSスタンプを。 20分間空気中のタイムスタンプを乾かします。ゆっくりと40秒のために金表面にスタンプを置き、金表面とスタンプ間の良好な接触があることを確認してください。過剰な圧力は必要ありません。エタノールで表面をリンスし、N 2気流下でそれを乾燥させてください。 </li><li> ° Cパターンの24.5で3時間灌流室（電子顕微鏡の科学）を用いて、P -セレクチン溶液（DPBS 15μg/ mLの）P -セレクチンとの残りの領域内の基板をインキュベートする。大量のDPBSで表面を洗浄します。 </li><li>埋め戻しの非特異的相互作用をブロックするために1時間BSA（DPBSで1 mg / mL）で表面。大量のDPBSで表面を洗浄します。 </li></ol> 1.2。フローチャンバーで実験をローリング細胞。 <ol><li>長方形のフローチャンバーにパターン化表面上にHL60細胞（〜10 5細胞/ mL）（; 幅 w = 1.0センチメートル、長さ= 6センチメートル、Glycotech、株式会社高さh = 0.0127センチメートル）の懸濁液を流す24.5℃で0.5 DYN / cm 2と （〜0.05 Pa）の周りの対応するせん断応力と、75μL/ minの流量を生成するためのシリンジポンプ（世界の精密機器、SP230IW）を使用してください。 τ平面ポアズイユ流の方程式体積流量Qは、μは動粘度（0.001002 PaのS）であるτは=6μQ/wh2を 、使用して、せん断応力を計算し、wはフローチャンバーの幅であり、hは高さです。フローチャンバー。 </li><li> 300の期間については、通常、1秒あたり1フレームの速度で、4を用いて接着剤P -セレクチン基質との相互作用をローリングHL60細胞を記録するために設置されたカメラ（アンドールIXON 885）×目標と倒立顕微鏡（ニコンTE2000 - U）を使用してくださいS.それぞれのせん断応力の大きさとパターンの傾斜角の3つの独立した実験を、実行します。各実験から得られた平均値の平均と標準偏差として存在するデータ。 </li><li>データ解析。 パターニングされた線のエッジに沿ってトラックを生成するフリーウェア8を追跡粒子を利用したカスタマイズされたMatlabの（MathWorks社）プログラムによって画像のシーケンスを分析する。 P -セレクチンバンドの終わりまで拡張するトラックを選択し、2本の直線の線分が備わっています - 流れに沿った一つのパターンのエッジに揃え、他。これら2つのセグメントは、次にエッジで速度を圧延し、平野地域で速度を圧延、エッジ追跡の長さを計算するために使用されています。 </li></ol> 2。代表的な結果： <img src="/files/ftp_upload/2640/2640fig1.jpg" alt="図1" /> 図（A）は 4 ×の対物レンズを用いて接着剤P -セレクチン基質とHL60ローリング相互作用のビデオから変換した顕微鏡画像の一つを示しています。明るい領域と暗い領域はそれぞれ、P -セレクチン受容体とPEGの地域に対応しています。 図（B）は、カスタマイズされたMatlabのプログラムを使用して得られたトラックを示しています。エッジの傾斜角度は10 °であり、せん断応力は、0.5 DYN / cm 2であった。エッジ追跡の長さ、L E、変位、D、およびエッジ上やバンド内の圧延速度は、それぞれV、EとV のp、、 図（C - 1）に記載されている。 図（C - 2）を示しエッジ追跡の長さの分布（記録されたセルの数）。インセットは、L Eの平均値とエッジの圧延速度（V、E）を示し、傾斜角のバンド（V P）内部のα= 10 °および0.5 DYN / cm 2と周りの流体せん断応力の大きさ。エラーバーは、各条件の1つの標準偏差、n = 3の複製実験を（3回の反復面）を表します。

<ol>
	<li>Karnik, R., Hong, S., Zhang, H., Mei, Y., Anderson, D. G., Karp, J. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomechanical+control+of+cell+rolling+in+two+dimensions+through+surface+Patterning+of+receptors.">Nanomechanical control of cell rolling in two dimensions through surface Patterning of receptors.</a> Nano Lett. 8 (4), 1153-1158 (2008).</li><li>Lee, C. H., Bose, S., Van Vliet, K. J., Karp, J. M., Karnik, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+Lateral+Displacement+of+HL60+Cells+Rolling+on+Asymmetric+P-selectin+Patterns.">Examining Lateral Displacement of HL60 Cells Rolling on Asymmetric P-selectin Patterns.</a> Langmuir. 27 (1), 240-249 (2011).</li><li>Norman, K. E., Moore, K. L., McEver, R. P., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+rolling+in-vivo+is+mediated+by+p-selectin+glycoprotein+ligand-1.">Leukocyte rolling in-vivo is mediated by p-selectin glycoprotein ligand-1.</a> Blood. 86 (12), 4417-4421 (1995).</li><li>Bernard, A., Delamarche, E., Schmid, H., Michel, B., Bosshard, H. R., Biebuyck, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+patterns+of+proteins.">Printing patterns of proteins.</a> Langmuir. 14 (9), 2225-2229 (1998).</li><li>James, C. D., Davis, R. C., Kam, L., Craighead, H. G., Isaacson, M., Turner, J. N., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+protein+layers+on+solid+substrates+by+thin+stamp+microcontact+printing.">Patterned protein layers on solid substrates by thin stamp microcontact printing.</a> Langmuir. 14 (4), 741-744 (1998).</li><li>Mrksich, M., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+self-assembled+monolayers+to+understand+the+interactions+of+man-made+surfaces+with+proteins+and+cells.">Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells.</a> Annual Review of Biophysics and Biomolecular Structure. 25, 55-78 (1996).</li><li>Tan, J. L., Tien, J., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+printing+of+proteins+on+mixed+self-assembled+monolayers.">Microcontact printing of proteins on mixed self-assembled monolayers.</a> Langmuir. 18 (2), 519-523 (2002).</li><li>Lee, D., King, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microcontact+Printing+of+P-Selectin+Increases+the+Rate+of+Neutrophil+Recruitment+Under+Shear+Flow.">Microcontact Printing of P-Selectin Increases the Rate of Neutrophil Recruitment Under Shear Flow.</a> Biotechnology Progress. 24 (5), 1052-1059 .</li><li>Greenberg, A. W., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+mediated+by+differential+rolling+adhesion.">Cell separation mediated by differential rolling adhesion.</a> Biotechnol. Bioeng. 73 (2), 111-124 (2001).</li><li>Higuchi, A., Tsukamoto, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separation+of+hepatocytes+and+fibroblasts+through+surface-modified+polyurethane+membranes.">Cell separation of hepatocytes and fibroblasts through surface-modified polyurethane membranes.</a> J. Biomed. Mater. Res. Part A. 71A (3), 470-479 (2004).</li><li>Alexeev, A., Verberg, R., Balazs, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterned+surfaces+segregate+compliant+microcapsules.">Patterned surfaces segregate compliant microcapsules.</a> Langmuir. 23 (3), 983-987 (2007).</li></ol>

利害の衝突は宣言されません。

我々は、マイクロコンタクトプリンティング2を用いて作製した非対称受容体柄の表面にセルローリング軌跡を調べるために、プロトコルを記述している。 PEGとP -セレクチンの領域の間に明確なコントラストを示すパターン化表面の光学顕微鏡像は、スタンピングが成功したかどうかを確認するために使用することができます。シャープ、ストレートエッジは刻印がよく実行されるときに観察することができます。ハードスタンプを押すと、パターンの精度を制限するタイムスタンプの変形が発生する可能性があります。インクの濃度が高すぎるまたはタイムスタンプが長すぎるときに波状の縁を得ることができる。スタンプと表面の間の接触不良は、スタンプのサイズは（&lt;0.5 cm 2）の小さすぎる場合、不均一なPEGのパターンにつながる。非新鮮なインク液ため、P -セレクチンを不動態化能力の低下のPEG -パターン化された領域の上を転動する細胞になることがあります。実際の傾斜角は、表面と相互作用していない自由流動性細胞のパターンと軌跡の画像から計算することができます。実験は、ここにパターンのエッジに沿ってイベントを転がり、個々のセルについての利回りの情報を説明しますが、自動的に別のパターンの1つのパターンにデタッチと再アタッチ細胞を追跡することは困難になります。パターンの間隔の変更、例えば、複数のエッジを検出した単セルのトラッキングを有効にすることができます。この能力は、単一細胞レベルでの異なる圧延特徴を有する細胞を区別するための高解像度を有効にすることができます。挙動を転がりセルがセル上にリガンドに依存するため、我々はそのような実験は9月12日、彼らのローリング動作3の違いに基づいて、異なる細胞表現型を分離するために適切なパターンの設計を可能にするかもしれないという仮説を立てた。この作品は、このように細胞のリガンドおよびパターン非対称受容体間の相互作用に基づく細胞の分離および分析用機器の設計に役立ちます。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human promyelocytic leukemia cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CCL-240</td>
<td>HL60 cells</td>
</tr>
<tr>
<td>Gold-coated glass slides</td>
<td>&nbsp;</td>
<td>EMF</td>
<td>TA134</td>
<td>Gold slides</td>
</tr>
<tr>
<td>(1-Mercaptoundec-11-yl)tetra(ethylene glycol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>674508</td>
<td>PEG</td>
</tr>
<tr>
<td>Recombinant human P-selectin</td>
<td>&nbsp;</td>
<td>R&amp;D Systems Inc.</td>
<td>ADP3-050</td>
<td>P-selectin</td>
</tr>
<tr>
<td>Bovine serum albumin</td>
<td>&nbsp;</td>
<td>Rockland Immunochemicals, Inc.</td>
<td>BSA-50</td>
<td>BSA</td>
</tr>
<tr>
<td>Dulbecco's phosphate buffered saline </td>
<td>&nbsp;</td>
<td>Mediatech Inc.</td>
<td>21-030</td>
<td>DPBS</td>
</tr>
<tr>
<td>Sulfuric acid </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>339741</td>
<td>&nbsp;</td>
<tr>
<td>Hydrogen peroxide</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>316989</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

studying cell rolling trajectories on asymmetric receptor patterns

非対称受容体パターン上を転動することによりフロー流に直交するセルの横方向変位は、セル1のラベルフリーの分離および分析用の新しいデバイスの開発のための機会を提供します。このようなデバイスは、連続フロー分離、または異なる細胞表現型または受容体の発現のレベルを区別するために接着を調節する受容体のパターンの横変位を使用することができます。受容体-パターニング基板上に軌跡を圧延細胞の性質を理解することは、基板とそのようなデバイスの設計のエンジニアリングが必要である。 ここでは、セルローリング接着2をサポートしている非対称の受容体のパターンでセルローリング軌道を研究するためのプロトコルを示す。 P -セレクチン受容体の明確に定義された、ミクロンスケールのパターンは、フローチャンバー内に組み込まれた金被覆スライド上にマイクロコンタクトプリントを作製した。 PSGL - 1リガンド3を発現HL60細胞がパターン化された行のフィールドを越え流れと逆の明視野顕微鏡で視覚化した。細胞は横方向のたわみ1で、その結果、パターンの傾斜縁部に沿ってロールバックして追跡。各セルは、一般的に、パターンのエッジに沿って一定の距離（エッジ追跡の長さとして定義される）のためにロール端から剥離し、下流のパターンに再接続。この剥離が困難フローチャンバーに終了するには、入り口から細胞の全体の軌跡を追跡することになりますが、粒子追跡のソフトウェアは、それらが単一の受容体に移動された時に細胞のローリング軌跡を分析し、生成するために使用されていました柄のライン。軌道は、セルの圧延速度と異なるパターンの各セルのエッジ追跡の長さの分布を得るために検討した。 このプロトコルは、受容体のパターンでセルローリング軌跡を定量化し、パターンの角とせん断応力などのこれらの工学的パラメータを関連づけるのに便利です。そのようなデータは、ラベルフリー細胞の分離および分析用マイクロ流体デバイスの設計に有効になります。

Watch this Scientific Journal Video about Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns at JoVE.com

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

我々は、非対称受容体柄の基板上にセルローリング軌跡を観察し、分析するためのプロトコルについて説明します。結果のデータは、ラベルフリー細胞分離と解析のための受容体柄の基板のエンジニアリングするのに便利です。

非対称受容体のパターンでセルローリング軌道を勉強

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

studying-cell-rolling-trajectories-on-asymmetric-receptor-patterns

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K ビュー.  MIT - Massachusetts Institute of Technology. 我々は、非対称受容体柄の基板上にセルローリング軌跡を観察し、分析するためのプロトコルについて説明します。結果のデータは、ラベルフリー細胞分離と解析のための受容体柄の基板のエンジニアリングするのに便利です。

ビデオ: Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

物質 - 細胞相互作用研究ナノポーラス金パターンの微細加工

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

生物工学

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

の体系的な分析<em&gt;体外</emマルチウェルプレートマイクロ流体システムを使用して&gt;セルローリング

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

はSiO：フォトリソグラフィによって画定されたパリレン-C上の細胞のパターニング<sub&gt; 2</sub&gt;基板

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

タンパク質分析のための継続的な認識パターンを生成する電子舌

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

蛍光生体膜フォースプローブ：単一細胞上の受容体 - リガンド速度論の同時定量との結合によって誘導される細胞内シグナル伝達

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

細胞内ターゲティングアプリケーションのための固体脂質ナノ粒子（SLNs）

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

相関顕微鏡を用いて、無傷の細胞における上皮成長因子受容体の化学量論を学びます

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

脳癌と神経変性疾患を研究するためのヒト神経オルガノイド

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

高分子表面上の吸着タンパク質層におけるトール様受容体媒介NF-kB/AP-1シグナル伝達を調べるマクロファージレポーター細胞アッセイ

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

マトリックス細胞代謝クロストークを研究するためのヒト3D細胞外マトリックス-脂肪細胞培養モデル

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...