Research related to this paper and the development of the fBFP technology in the Zhu lab were supported by NIH grants AI044902, AI077343, AI038282, HL093723, HL091020, GM096187, and TW008753. We thank Evan Evans for inventing this empowering experimental tool, and members of the Evans lab, Andrew Leung, Koji Kinoshita, Wesley Wong, and Ken Halvorsen, for helping us to build the BFP. We also thank other Zhu lab members, Fang Kong, Chenghao Ge and Kaitao Li, for their helps in the instrumentation development.

<ol>
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The authors have nothing to disclose.

成功fBFP実験は、いくつかの重要な考慮事項を必要とします。まず、力計算のために、マイクロピペット、RBC信頼性があるとし、プローブビーズはできるだけ同軸の近くに位置合わせされるべきです。 RBCとピペットとの間の摩擦が無視できるように、ピペット内のRBCの突起は、約1プローブピペット径でなければなりません。典型的なヒトRBCのために、最適なピペットの直径は、式1 17,30</su...

細胞-細胞及び細胞-細胞外マトリックス（ECM）接着は、細胞表面受容体、ECMタンパク質、および/ ​​または脂質の1との間の結合によって媒介されます。結合は、細胞が機能構造1を形成し、同様に認識し、通信し、環境1-3に対応することができます。可溶性タンパク質（ 例えば 、サイトカインおよび成長因子）、3次元（3D）から結合する細胞表面受容体への液相とは異なり、細胞接着受容体は、分子拘束二つの対向面をブリッジする狭い接合ギャップを横切るそのリガンドとの結合を形成します2次元（2D）インターフェース4-7の拡散。一般的に、伝統的な結合アッセイ（ 例えば 、表面プラズモン共鳴またはSPR）によって測定された3次元動態とは対照的に、2D速度は、原子間力顕微鏡（AFM）8-10のような特殊な技術を用いて定量化されなければならない、チャンバ11,12を流れ、マイクロピペット13,14、光ピンセット15と生体膜力プローブ（BFP）16-21。 単に携帯凝集のための物理的結合を提供するよりも、接着分子は、その周囲と通信するセルのシグナル伝達機構の主要な構成要素です。接着分子のリガンド係合は、細胞内シグナル伝達とどのように初期信号が細胞内に伝達されるがどのように開始するかを理解する上で関心が高まっています。直感的には、受容体 - リガンド結合の性質は、それが誘導信号に影響を与えることができます。しかし、それがために、例えば 、それらの多くの制限、貧しい時間分解能と空間分解能の完全な欠如の生化学的ア ​​ッセイの伝統的なアンサンブルを使用して外の相互作用および細胞内シグナル伝達事象の間の機械的な関係を分析することは困難です。両方の生物物理学的（2D受容体 - リガンド結合反応速度）を可能にする既存の方法とライブの生化学（シグナリング）の観察細胞は、調整可能な剛性22、エラストマーピラーアレイ23と蛍光能力24〜26を組み込んだフローチャンバー/マイクロ流体デバイスの基材が挙げられます。しかし、シグナル伝達および受容体 - リガンド結合の読み出しは、それが困難なシグナル伝達事象との結合特性の時間的、空間的な関係を分析すること、（異なる方法によって、ほとんどの場合）別途入手する必要があります。 従来のBFPは、高時空間分解能17と超力分光法です。これは、単一分子の2D速度、機械的特性およびコンフォメーション変化14,16,19-21,27-29の測定を可能にする、力センサのような可撓性赤血球（RBC）を使用します。 BFP（fBFP）系蛍光イメージングは​​、単一分子スケールでのシグナリング結合誘発される細胞での受容体 - リガンド結合反応速度を相関させます。この設定では、 その場での細胞内の表面mechaniのコンテキストでの活動に信号を送ります的刺激は、T-27細胞で観察されました。 fBFPは用途が広く、他のセル内の他の分子によって媒介される細胞接着およびシグナル伝達の研究のために使用することができます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="4">Table 1: Reagents/Equipment</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Monobasic Monohydrate (NaH2PO4&#8226;H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>Phosphate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Carbonate/bicarbonate buffer preparation</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>N2-5% buffer preparation</td>
		</tr>
		<tr>
			<td>MAL-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5002-1</td>
			<td>Bead functionalization</td>
		</tr>
		<tr>
			<td>Biotin-PEG3500-NHS</td>
			<td>JenKem</td>
			<td>A5026-1</td>
			<td>RBC biotinylation</td>
		</tr>
		<tr>
			<td>Nystatin</td>
			<td>Sigma-Aldrich</td>
			<td>N6261</td>
			<td>RBC osmolarity adjustment</td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide (NH4OH)</td>
			<td>Sigma-Aldrich</td>
			<td>A-6899</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>BDH</td>
			<td>67-56-1</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide (H2O2)</td>
			<td>J. T. Barker</td>
			<td>Jan-86</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>Acetic Acid (Glacial)</td>
			<td>Sigma-Aldrich</td>
			<td>ARK2183</td>
			<td>Glass bead silanization</td>
		</tr>
		<tr>
			<td>3-MERCAPTOPROPYLTRIMETHOXYSILANE(MPTMS)</td>
			<td>Uct Specialties, llc</td>
			<td>4420-74-0</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Borosilicate Glass beads</td>
			<td>Distrilab Particle Technology</td>
			<td>9002</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>Streptavidin&#8722;Maleimide</td>
			<td>Sigma-Aldrich</td>
			<td>S9415</td>
			<td>Glass bead functionalization</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A0336</td>
			<td>Ligand functionalizing</td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F-1201</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td>Intracellular calcium fluorescence dye loading</td>
		</tr>
		<tr>
			<td>Quantibrite PE Beads</td>
			<td>BD Biosciences</td>
			<td>340495</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td>BD LSR II</td>
			<td>Density quantification</td>
		</tr>
		<tr>
			<td>Capillary Tube 0.7-1.0mm x 30&#34;</td>
			<td>Kimble Chase</td>
			<td>46485-1</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>sutter instrument</td>
			<td>P-97</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Pipette microforce</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Micropipette making</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Fisher Scientific</td>
			<td>BP2629-1</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-G</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micro-injector</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>1ml Syringe</td>
			<td>BD&#160;</td>
			<td>309602</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Narishige</td>
			<td>HI-7</td>
			<td>Chamber assembly</td>
		</tr>
		<tr>
			<td>Home-designed mechanical parts and adaptors fabrications using CNC machining.&#160;</td>
			<td>Biophysics Instrument</td>
			<td>All parts are customized according to the CAD designs.</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Microscope (TiE inverted)</td>
			<td>Nikon</td>
			<td>MEA53100</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72mm, Spg)</td>
			<td>Nikon</td>
			<td>MRH00401</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Camera, GE680, 640x480, GigE, 1/3&#34; CCD, mono</td>
			<td>Graftek Imaging</td>
			<td>02-2020C</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Prosilica GC1290 - ICX445, 1/3&#34;, C-Mount, 1280x960, Mono., CCD, 12 Bit ADC</td>
			<td>Graftek Imaging</td>
			<td>02-2185A</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Manual submicron probehead with high resolution remote control</td>
			<td>Karl Suss</td>
			<td>PH400</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Anti-vibration table (5&#8217; x 3&#8217;)</td>
			<td>TMC</td>
			<td>77049089</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>3D manual translational stage</td>
			<td>Newport</td>
			<td>462-XYZ-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SolidWorks 3D CAD software</td>
			<td>SOLIDWORKS Corp.</td>
			<td>Version 2012 SP5</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>LabVIEW software</td>
			<td>National Instruments</td>
			<td>Version 2009</td>
			<td>BFP system, BFP program</td>
		</tr>
		<tr>
			<td>3D piezo translational stage</td>
			<td>Physik Instrumente</td>
			<td>M-105.3P</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Linear piezo accuator</td>
			<td>Physik Instrumente</td>
			<td>P-753.1CD</td>
			<td>BFP system</td>
		</tr>
		<tr>
			<td>Micromanager software</td>
			<td></td>
			<td>Version 1.4</td>
			<td>fBFP system, fluorescence imaging program</td>
		</tr>
		<tr>
			<td>Dual Cam (DC-2)</td>
			<td>Photometrics</td>
			<td>77054724</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Dual Cam emission filter (T565LPXR)</td>
			<td>Photometrics</td>
			<td>77054725</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Fluorescence Camera</td>
			<td>Hamamatsu</td>
			<td>ORCA-R2 C10600-10B</td>
			<td>fBFP system</td>
		</tr>
		<tr>
			<td>Plastic paraffin film (Parafilm)</td>
			<td>Bemis Company, Inc</td>
			<td>PM996</td>
			<td>bottle sealing</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Table 2: Buffer solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate/bicarbonate buffer (pH 8.5)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate (Na2CO3)</td>
			<td>8.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>10.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer (pH 6.5-6.8)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaPhosphate monobasic&#160;&#160;&#160;NaH2PO4&#8226;H2O</td>
			<td>27.6g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. NaPhosphate dibasic&#160;&#160; Na2HPO4</td>
			<td>28.4g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2-5% buffer (pH 7.2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>20.77g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>2.38g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>0.13g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anhy. Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td>0.71g/L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>9.70g/L</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell

膜受容体 - リガンド相互作用は、多くの細胞機能を媒介します。これらの分子の相互作用によって引き起こさ結合動態および下流のシグナル伝達は、おそらく結合し、場所を取るシグナル伝達する機械的な環境の影響を受けています。最近の研究では、機械的な力により、抗原認識を調節し、T細胞受容体（TCR）をトリガすることができることを実証しました。これは、我々が開発した新技術によって可能になったし、蛍光顕微鏡で単一分子力分光法を組み合わせた蛍光生体膜力プローブ（fBFPを）、と呼ばれました。敏感な力センサー、高速度カメラとリアルタイムイメージング追跡技術として、超ソフトヒト赤血球を用いて、fBFPは約3 nmおよび〜0.5秒で約1 PN（10 -12 N）であります力、空間的および時間的分解能。 fBFPで、1は正確に力調節の下で単一の受容体 - リガンド結合反応速度と同時に、画像結合トリガー細胞内CALを測定することができます単一の生細胞上でシグナリングcium。この新技術は、機械的な規制の下に他のセルにおける他の膜受容体 - リガンド相互作用およびシグナル伝達を研究するために使用することができます。

BFP技術は1995 17エバンス研究所によって開発された。このpicoforceツールは広く、そのリガンド16,19,20と相互作用する接着分子の二次元の動態を解析するように、表面上に固定化されたタンパク質の相互作用を測定するために使用されています30、分子21,29弾性を測定するため、およびタンパク質コンホメーション変化21を決定します。 fBF...

Watch this Scientific Journal Video about Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell at JoVE

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

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 ...

fluorescence-biomembrane-force-probe-concurrent-quantitation-receptor

Research

JoVE Journal

Bioengineering

12.3K ビュー.  Georgia Institute of Technology. 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.

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