This study was supported by NIH grants R01HL091020, R01HL093723, R01AI077343, and R01GM096187.

1. RBCs isolation from the whole blood
<ol>
 <li>Prepare EAS-45 solutions. Weigh up all ingredients from Table I and dissolve in 100-200ml of DI water. Add water to make 1000ml solution and adjust pH to 8.0. Filter and aliquot by 50ml. Freeze at -20&deg;C for storage.</li>
</ol>
Note: Step 1.2 should be performed by a trained medical professional such as a nurse, with an Institutional Review Board approved protocol.
<ol start="2">
 <li>Draw 3-5ml of blood from the median cub...

<ol>
	<li>Chesla, S. E., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+two-dimensional+receptor-ligand+binding+kinetics+by+micropipette.">Measuring two-dimensional receptor-ligand binding kinetics by micropipette.</a> Biophys. J. 75, 1553-1572 (1998).</li><li>Chesla, S. E., Li, P., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+anchor+influences+ligand+binding+two-dimensional+kinetic+rates+and+three-dimensional+affinity+of+FcgammaRIII+(CD16).">The membrane anchor influences ligand binding two-dimensional kinetic rates and three-dimensional affinity of FcgammaRIII (CD16).</a> J. Biol. Chem. 275, 10235-10246 (2000).</li><li>Williams, T. E., Nagarajan, S., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+and+independent+binding+of+Fcgamma+receptors+IIa+and+IIIb+to+surface-bound+IgG.">Concurrent and independent binding of Fcgamma receptors IIa and IIIb to surface-bound IgG.</a> Biophys. J. 79, 1867-1875 (2000).</li><li>Williams, T. E., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+binding+to+multiple+ligands:+kinetic+rates+of+CD16b+for+membrane-bound+IgG1+and+IgG2.">Concurrent binding to multiple ligands: kinetic rates of CD16b for membrane-bound IgG1 and IgG2.</a> Biophys. J. 79, 1858-1866 (2000).</li><li>Williams, T. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+concurrent+binding+of+multiple+molecular+species+in+cell+adhesion.">Modeling concurrent binding of multiple molecular species in cell adhesion.</a> Biophys. J. 79, 1850-1857 (2000).</li><li>Downey, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+leukocytes+in+capillaries:+role+of+cell+size+and+deformability.">Retention of leukocytes in capillaries: role of cell size and deformability.</a> J. Appl. 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A., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+force+dependence+of+two-dimensional+receptor-ligand+binding+affinity+by+centrifugation.">Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.</a> Biophys. J. 74, 492-513 (1998).</li><li>Li, P., Selvaraj, P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+competition+binding+between+soluble+and+membrane-bound+ligands+for+cell+surface+receptors.">Analysis of competition binding between soluble and membrane-bound ligands for cell surface receptors.</a> Biophys. J. 77, 3394-3406 (1999).</li><li>Long, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probabilistic+modeling+of+rosette+formation.">Probabilistic modeling of rosette formation.</a> Biophys. J. 91, 352-363 (2006).</li><li>Lou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-enhanced+adhesion+regulated+by+a+selectin+interdomain+hinge.">Flow-enhanced adhesion regulated by a selectin interdomain hinge.</a> J. Cell. Biol. 174, 1107-1117 (2006).</li><li>Yago, T., Zarnitsyna, V. I., Klopocki, A. G., McEver, R. P., Zhu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+governs+flow-enhanced+cell+tethering+through+L-selectin+at+threshold+shear.">Transport governs flow-enhanced cell tethering through L-selectin at threshold shear.</a> Biophys. J. 92, 330-342 (2007).</li><li>Evans, E., Berk, D., Leung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detachment+of+agglutinin-bonded+red+blood+cells.+I.+Forces+to+rupture+molecular-point+attachments.">Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular-point attachments.</a> Biophys. J. 59, 838-848 (1991).</li><li>Zarnitsyna, V. 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No conflicts of interest declared.

To successfully use the micropipette adhesion frequency assay one should consider several critical steps. First, make sure to record the specific interaction for the receptor-ligand system of interest. Nonspecific control measurements (cf. Fig. 3, 4) ensure the specificity. Ideally, nonspecific adhesion probabilities should be below 0.05 for all contact time durations and to have a significant difference between the specific and nonspecific adhesion probabilities for each time point. Different methods could be used to co...

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company</th>
<th>Catalogue #</th>
<th>Comments</th>
</tr>
<tr>
<td>10x PBS</td>
<td>BioWhittaker</td>
<td>17-517Q</td>
<td>Dilute to 1x with deionized water prior to use</td>
</tr>
<tr>
<td>Vacutainer EDTA </td>
<td>BD</td>
<td>366643</td>
<td>RBCs isolation</td>
</tr>
<tr>
<td>10ML PK100</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Histopaque 1077</td>
<td>Sigma-Aldrich</td>
<td>10771</td>
<td>RBCs isolation</td>
</tr>
<tr>
<td>Adenine</td>
<td>Sigma-Aldrich</td>
<td>A2786</td>
<td>EAS-45 preparation</td>
</tr>
<tr>
<td>D-glucose (dextrose)</td>
<td>Sigma-Aldrich</td>
<td>G7528</td>
<td>EAS-45 preparation</td>
</tr>
<tr>
<td>D-Mannitol</td>
<td>Sigma-Aldrich</td>
<td>6360</td>
<td>EAS-45 preparation</td>
</tr>
<tr>
<td>Sodium Chloride (NaCl)</td>
<td>Sigma-Aldrich</td>
<td>S7653</td>
<td>EAS-45 preparation</td>
</tr>
<tr>
<td>Sodium Phosphate, Dibasic (Na&#x2082;HPO&#x2084;)</td>
<td>Fisher Scientific</td>
<td>S374</td>
<td>EAS-45 preparation</td>
</tr>
<tr>
<td>L-glutamine</td>
<td>Sigma-Aldrich</td>
<td>G5763</td>
<td>EAS-45 preparation</td>
</tr>
<tr>
<td>Biotin-X-NHS</td>
<td>Calbiochem</td>
<td>203188</td>
<td>RBCs biotinylation</td>
</tr>
<tr>
<td>Dimethylformamide (DMF)</td>
<td>Thermo Scientific</td>
<td>20673</td>
<td>RBCs biotinylation</td>
</tr>
<tr>
<td>Borate Buffer (0.1M)</td>
<td>Electron Microscopy Sciences</td>
<td>11455-90</td>
<td>RBCs biotinylation</td>
</tr>
<tr>
<td>Streptavidin</td>
<td>Thermo Scientific</td>
<td>21125</td>
<td>Ligand functionalizing</td>
</tr>
<tr>
<td>BSA</td>
<td>Sigma-Aldrich</td>
<td>A0336</td>
<td>Ligand functionalizing</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 Immunocytometry Systems</td>
<td>BD LSR II</td>
<td>Density quantification</td>
</tr>
<tr>
<td>Capillary Tube
0.7-1.0mm x 30&quot;</td>
<td>Kimble Glass Inc.</td>
<td>46485-1</td>
<td>Micropipette pulling</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>PE &alpha;-human CD11a 
Clone HI 111 </td>
<td>eBioscience</td>
<td>12-0119-71</td>
<td>Reagent for Fig.1</td>
</tr>
<tr>
<td>PE anti-human CD54 </td>
<td>eBioscience</td>
<td>12-0549</td>
<td>Reagent for Fig.1</td>
</tr>
<tr>
<td>Mouse IgG1 Isotype Control PE</td>
<td>eBioscience</td>
<td>12-4714</td>
<td>Reagent for Fig.1</td>
</tr>
<tr>
<td>hydraulic micromanipulator</td>
<td>Narishige</td>
<td>MO-303</td>
<td>Micropipette system</td>
</tr>
<tr>
<td>Mechanical manipulator</td>
<td>Newport</td>
<td>461-xyz-m, SM-13, DM-13</td>
<td>Micropipette system</td>
</tr>
<tr>
<td>piezoelectric translator</td>
<td>Physik Instrumente</td>
<td>P-840</td>
<td>Micropipette system</td>
</tr>
<tr>
<td>LabVIEW</td>
<td>National Instruments</td>
<td>Version 8.6</td>
<td>Micropipette system</td>
</tr>
<tr>
<td>DAQ board</td>
<td>National Instruments</td>
<td>USB-6008</td>
<td>Micropipette system</td>
</tr>
<tr>
<td>Optical table</td>
<td>Kinetics Systems</td>
<td>5200 Series</td>
<td>Micropipette system</td>
</tr>
</tbody>
</table>

adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Watch this Scientific Journal Video about Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface at JoVE.com

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

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14.9K 조회수.  Georgia Institute of Technology. 두 분자가 상호 작용하는 세포의 표면에 고정하는 경우 수용체 - 리간드 상호 작용 속도론을 측정하는 접착 주파수 분석이 설명되어 있습니다. 이 기계 기반의 분석은 수용체와 리간드를 상호 작용으로 micropipette - 압력 인간의 붉은 부착 센서와 같은 혈액 세포와 인테 αLβ2과 세포 유착 분자 - 1을 사용하여 exemplified입니다.