Name: fluorescence biomembrane force probe: concurrent quantitation of receptor-ligand kinetics and binding-induced intracellular signaling on a single cell
Uploaded: 2015-08-04T13:00:00
Description: 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

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.

이 프로토콜의 지침을 다음과 조지아 공대의 인간 연구 윤리위원회에 의해 승인되었습니다. 1. 인간의 적혈구 분리, 바이오 티 닐화 및 삼투압 조절 참고 : 임상 시험 심사위원회가 프로토콜을 승인 한 단계 1.1, 숙련 된 의료 전문가 등의 간호사가 수행해야합니다. <ol><li> 손가락 찌르기에서 혈액의 8-10 μL (한 방울)를 얻어 탄산염 / 중탄산염 버퍼 1 ㎖에...

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성공적인 fBFP 실험은 몇 가지 중요한 고려 사항을 수반한다. 힘의 계산이 신뢰할 수있는 위해 첫째, 마이크로 피펫, RBC, 프로브 비드가 가능한 동축에 가깝게 정렬되어야합니다. RBC 및 피펫 사이의 마찰을 무시할 수 있도록 피펫 내부 RBC의 투영 약 1 프로브 피펫 직경이어야한다. 전형적인 사람 RBC 들어, 최적 피펫 직경 17,30 식 1의 최적을 산출 2.0-2.4 ㎛ 인 것이다. 둘째, 힘 클램프 분석 및 ?...

세포 간 및 세포 - 대 - 세포 외 기질 (ECM)의 밀착성이 세포 표면 수용체, ECM 단백질, 및 / 또는 지질 1 간의 결합에 의해 매개된다. 바인딩은 세포의 기능을 형성 한 구조뿐만 아니라, 인식, 통신, 및 환경 1-3에 반응 할 수있다. 수용성 단백질 (예 : 사이토킨 및 성장 인자), 3 차원 (3D)에서 결합하는 세포 표면 수용체 상 유체 상 달리 세포 부착 수용체 분자 구속 두 대향면을 연결하는 좁은 접합부 갭에 걸쳐 그들의 리간드 결합을 형성 두 차원 (2D) 인터페이스 4-7에 확산. 일반적으로 기존의 결합 분석 (예를 들어, 표면 플라즈몬 공명 또는 SPR)에 의해 측정되는 3D 동역학 달리, 챔버 (11, 12)을 흘러, 예컨대 원 자간 력 현미경 (AFM) 8-10 같은 전문 기술을 정량 2D 동력학 가지고 마이크로 피펫 13, 14, 광핀셋 (15)과 생체막의 힘 프로브 (BFP) 16-21. 단지 세포 응집력 물리적 링크를 제공하는 이상은, 부착 분자는 그 주변과 통신하는 셀에 대한 시그널링 장치의 주요 구성 요소이다. 부착 분자의 리간드 결합은 세포 내 신호 전달 및 방법은 초기 신호가 상기 형질 도입 된 세포의 내부를 개시한다 방법 이해에 관심이 증대되고있다. 직관적으로, 수용체 - 리간드의 성질은 그것이 유도 된 신호에 영향을 미칠 수 바​​인딩. 그러나, 그 때문에 예를 들면 많은 한계, 불량한 시간 해상도와 공간 해상도의 완전한 결핍의 생화학 적 분석법 전통적인 앙상블을 사용 세포 상호 작용 및 세포 내 신호 전달 기전 이벤트 간의 관계를 해부하기가 어렵다. 라이브에 모두 생물 물리학 (역학을 결합 2D 수용체 - 리간드)을 허용 방법과 생화학 (신호) 관측을 기존세포는 형광 기능을 24-26로 통합 조정 강성 (22)의 기판 (23) 엘라스토머 기둥 배열과 유량 용기 / 미세 유체 장치를 포함한다. 그러나, 신호와 결합 수용체 리간드의 판독이 어려운 신호 이벤트와 결합 특성의 공간적 관계를 해부하고, (다른 방법으로 가장 자주) 개별적으로 얻을 수있다. 기존의 BFP는 높은 시공간 해상도 (17)와 고감도 힘 분광법이다. 이는 단일 분자 동력학 2D, 기계적 특성 및 구조적 변화 14,16,19-21,27-29의 측정을 가능하게 힘 센서로서가요 적혈구 (RBC)를 사용한다. 형광 이미징 기반 BFP (fBFP)는 단일 분자 규모에서 결합 트리거 세포 신호와 수용체 - 리간드 결합 반응 속도의 상관 관계. 표면 mechani의 맥락에서 현장 세포 신호 활동이 설정으로CAL 자극은 T-27 세포에서 관찰되었다. fBFP 만능이며 다른 셀들에있는 다른 분자들에 의해 매개되는 세포 부착 및 시그널링의 연구에 사용될 수있다.

<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

멤브레인 수용체 - 리간드 상호 작용은 다수의 세포 기능을 매개한다. 바인딩 역학 이러한 분자의 상호 작용에 의해 트리거 다운 스트림 신호는 가능성이 기계적인 환경에서 결합 및 신호 포획 장소에 의해 영향을받습니다. 최근 연구는 기계적인 힘에 의한 항원 인식을 조절하고 T 세포 수용체 (TCR)의 트리거링 할 것을 보여 주었다. 이 형광 현미경으로 단일 분자 힘 분광법을 결합한 새로운 우리가 개발 한 기술이라고 형광 생체막의 힘 프로브 (fBFP)에 의해 가능하게되었다. 민감한 힘 센서로서 고속 카메라와 실시간 영상 추적 기술을 매우 부드러운 인간 적혈구를 사용 fBFP 1 ~ PN (10-12 N) ~ 3nm 이상이며 ~ 0.5 밀리의 힘, 공간과 시간 해상도. fBFP으로, 하나는 정확하게 힘 조절에 따라 단일 수용체 - 리간드 결합 동력학과 동시​​에 이미지가 트리거 바인딩 세포 내 칼을 측정 할 수 있습니다cium 하나의 살아있는 세포에 신호. 이 새로운 기술은 기계적인 규제에서 다른 세포에서 다른 막 수용체 - 리간드 상호 작용과 신호 전달을 연구하는 데 사용할 수 있습니다.

BFP 기술 1995 17 에반스 실험실에 의해 개척되었다.이 picoforce 도구는 광범위하게, 그들의 리간드 16,19,20과 상호 작용하는 부착 분자의 이차원 동력학을 분석하기 위하여, 표면에 고정화 된 단백질의 상호 작용을 측정하기 위해 사용 된 30, 분자 탄성 21,29을 측정하고, 단백질의 구조적 (21)을 변경 결정합니다. 대한 fBFP, 추가되는 해당 소프트웨어 시스?...

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.

형광 생체막 포스 프로브 : 하나의 세포에서 수용체 - 리간드 속도론의 동시 정량 및 바인딩에 의한 세포 내 신호

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

Research

JoVE Journal

Bioengineering

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

RootChip를 사용하여 루트 환경의 신속한 조작과 Arabidopsis 루트 성장의 시간 저속 형광 이미징

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, ...

연구

생체공학

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

미세 유체 기술은 셀 변형능을 검사 할

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

스퀴지 기반의 조립 방법과 생체막 마이크로 어레이의 형성

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

수정 된 두 개의 광자 현미경에서 3D 궤도 추적 : 세포 내 소포의 추적에 응용

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

상용 현미경에서 라이브 세포막에 분자 확산 법률에 빠른 형광 이미징

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Surface Potential Measurement of Bacteria Using Kelvin Probe Force Microscopy

켈빈 프로브 힘 현미경을 사용하여 박테리아의 표면 전위 측정

Surface potential is a commonly overlooked physical characteristic that plays a dominant role in the adhesion of microorganisms to substrate surfaces. ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

용매를 이용한 지질 이중층 (SALB) 방법에 의해 생체막 제작

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

평면에 단일 분자 형광 현미경은 이중층 지원

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...