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.

A successful fBFP experiment entails a few critical considerations. First, for the force calculation to be reliable, the micropipette, the RBC, and the probe bead should be aligned as close to coaxial as possible. The projection of the RBC inside the pipette should be about one probe pipette diameter so that the friction between the RBC and the pipette is negligible. For a typical human RBC, the optimal pipette diameter is 2.0-2.4 &#181;m, which yields a best fit of Equation 117,30. Second, to ensure measureme...

Cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is mediated by binding between cell surface receptors, ECM proteins, and/or lipids1. Binding allows cells to form functional structures1, as well as recognize, communicate, and react to the environment1-3. Unlike soluble proteins (e.g., cytokines and growth factors) that bind from a three-dimensional (3D) fluid phase onto the cell surface receptors, cell adhesion receptors form bonds with their ligands across a narrow junctional gap to bridge two opposing surfaces that constrain molecular diffusion in a two dimensional (2D) interface4-7. In contrast to 3D kinetics that are commonly measured by traditional binding assays (e.g., surface plasmon resonance or SPR), 2D kinetics have to be quantified with specialized techniques such as atomic force microscopy (AFM)8-10, flow chamber11,12, micropipette13,14, optical tweezers15 and biomembrane force probe (BFP)16-21.
More than merely providing physical linkage for cellular cohesion, adhesion molecules are a major component of the signaling machinery for the cell to communicate with its surroundings. There has been increasing interest in understanding how ligand engagement of adhesion molecules initiates intracellular signaling and how the initial signal is transduced inside the cell. Intuitively, properties of receptor-ligand binding can impact the signals it induces. However, it is difficult to dissect mechanistic relationships between the extracellular interaction and intracellular signaling events using traditional ensemble of biochemical assays because of their many limitations, e.g., a poor temporal resolution and the complete lack of spatial resolution. Existing methods that allow both biophysical (2D receptor-ligand binding kinetics) and biochemical (signaling) observations on live cells include substrates of tunable rigidity22, elastomer pillar arrays23 and flow chamber/microfluidic devices incorporated with fluorescence capability24-26. However, readouts of signaling and receptor-ligand binding have to be obtained separately (most often by different methods), making it difficult to dissect temporal and spatial relations of bond characteristics with signaling events.
Conventional BFP is an ultrasensitive force spectroscopy with high spatiotemporal resolution17. It uses a flexible red blood cell (RBC) as a force sensor, enabling measurement of single-molecule 2D kinetics, mechanical properties and conformational changes14,16,19-21,27-29. A fluorescent imaging based BFP (fBFP) correlates the receptor-ligand binding kinetics with the binding-triggered cell signaling at single-molecule scale. With this setup, in situ cell signaling activities in the context of surface mechanical stimulation was observed in T-cells27. The fBFP is versatile and can be used for studies of cell adhesion and signaling mediated by other molecules in other cells.

<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

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.

The BFP technique was pioneered by the Evans laboratory in 1995 17. This picoforce tool has been extensively used to measure interactions of proteins immobilized on surfaces, so as to analyze two-dimensional kinetics of adhesion molecules interacting with their ligands16,19,20,30, to measure molecular elasticity21,29, and to determine protein conformational changes21. For an fBFP, an additional set of epi-fluorescence-related devices with the corresponding software system (<str...

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.

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 Views.  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.

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