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 cubital vein into a 10ml tube containing EDTA and gently mix the blood with EDTA immediately and thoroughly to avoid clotting.</li>
 <li>Process blood sample as soon as possible. All the following steps except centrifugation should be done under the hood to keep the preparation sterile. Transfer the blood to a 50ml centrifuge tube, add 10ml of cold and sterile Histopaque 1077 and centrifuge for 5min @ 1000rpm, 4&deg;C. Repeat twice.</li>
 <li>Add 10ml cold and sterile PBS, centrifuge for 5min @ 1000rpm, 4&deg;C. Remove the supernatant. Repeat 4 times (Total of 5 times).</li>
 <li>Wash with sterile EAS-45 (5min @ 1000rpm, 4&deg;C) twice. During last washing move RBCs into a new sterile 15ml tube.</li>
 <li>Resuspend RBCs in10ml EAS-45.</li>
</ol>
2. RBCs biotinylation
<ol>
 <li>Take the tube with RBCs from step 1. Remove all supernatant after centrifuging for 5min @ 1000rpm, 4&deg;C. Put 10&mu;l of RBCs pellet to each of several vials and add corresponding amount of PBS to each vial (see Table II for example of preparation of six different RBCs biotinylation concentrations).</li>
 <li>Make fresh Biotin-X-NHS/DMF 1:10, 1:100 dilutions according to Table II.</li>
 <li>Add 10&mu;l of 0.1M borate buffer to each vial.</li>
 <li>Add calculated amount of biotin solution to each vial (see Table II as example) and vortex immediately.</li>
 <li>Place each RBC vial inside a 50ml conical tube and incubate on rotator for 30min at room temperature.</li>
 <li>Wash each vial 3 times with 800&mu;l of EAS-45 for 2min @ 2000rpm.</li>
 <li>Add 100&mu;l of EAS-45 to each vial and store at 4&deg;C. In each 1&mu;l of the final solution now should be &tilde;1mln of RBCs.</li>
</ol>
3. Functionalizing the biotin-linked ligands* on RBCs
<ol>
 <li>Prepare 2mg/ml streptavidin solution according to manufacturer's instructions. Aliqouted solution may be stored at -20&deg;C.</li>
 <li>Mix an equal amount of RBCs from step 2.7 (10&mu;l) with streptavidin solution, vortex immediately and incubate on rotator for 30min at 4&deg;C.</li>
 <li>Wash 3 times with 500&mu;l of EAS-45 for 2min @ 2000rpm. Add 15&mu;l EAS-45/ 1% BSA for storage.</li>
 <li>Mix equal amount of RBCs from step 3.3 (10&mu;l) with 20&mu;g/ml ligand solution, vortex immediately and incubate on rotator for 30min at room temperature.</li>
 <li>Wash two times with 500&mu;l of EAS-45/ 1% BSA for 2min @ 2000rpm. Add 15&mu;l of EAS-45/1% BSA for storage.</li>
</ol>
*If your protein has no biotin link you can use one of the commercially available kits for protein biotinylation (for example, Thermo Scientific # 21955 EZ-Link Micro NHS-PEG4-Biotinylation Kit) or use biotinylated capturing antibodies as an intermediate step as shown in the video.
4. Quantification of receptor and ligand densities
<ol>
 <li>Incubate ligand-coated RBCs from step 3 and, in a different vial, receptor-bearing cells with saturating concentrations of respective mAbs for 30min at room temperature. Incubate in separate vials cells with irrelevant isotype-matched antibodies for control. If the primary antibodies are not fluorescently labeled, incubate with fluorescently-conjugated secondary antibodies according to manufacturer's instructions.</li>
 <li>Analyze samples prepared in step 4.1 by flow cytometery with corresponding fluorescent calibration beads. Calculate the densities as shown in Fig. 1.</li>
</ol>
5. Preparation for micropipette and cell chamber
<ol>
 <li>Pull micropipettes from capillary tubes using PN-30 Narishige' Magnetic Glass Microelectrode Horizontal Puller or Sutter Instruments Micropipette Puller.</li>
 <li>Use micromanipulator with microforge to adjust the tip of the pulled micropipette to a desired size (usually the required diameter ranges from 1-5&mu;m depending on the size of the cells to be used in the study).</li>
 <li>Prepare the cell chamber by cutting Microscope Cover Glass to the desired size. Seal the chamber using Mineral Oil from both sides to avoid medium evaporation to change the osmolarity during the experiment.</li>
 <li>Add the receptor- and ligand-bearing cells to the chamber.</li>
</ol>
6. Micropipette adhesion frequency assay
<ol>
 <li>Aspirate the interacting cells by respective pipettes and use computer-programmed piezoelectric translator to drive the RBC in and out of contact with the other cell with controlled contact area and time. Detect adhesion events by observing RBC elongation upon cell separation.</li>
 <li>Repeat the contact-retraction cycle 50-100 times for a given contact time. Record the observed adhesion events by adding "1" for adhesion or "0" for no adhesion in a column of Excel spreadsheet. You may use a recording device, e.g., digital media or videotape, for the microscopic images.</li>
 <li>Record the adhesion frequency versus contact time curve using at least three different cell pairs for each contact time to obtain a mean and SEM.</li>
 <li>Record the nonspecific binding curve for control by using RBCs coated with irrelevant ligands (e.g. BSA) and/or blocking the ligands or receptors using their specific functional blockade mAbs. Specific adhesion frequency at each contact time point can be calculated by removal of the nonspecific adhesion frequency1.</li>
</ol>
7. Data analysis
<ol>
 <li>Fit the specific adhesion frequency Pa versus contact time t data by a probabilistic model (Equation 1) that describes a second-order forward and first-order reverse, single-step interaction between a single species of receptors and a single species of ligands1: 
 <img alt="Equation 1" src="/files/ftp_upload/3519/3519eq1.jpg" /> 
 where Ka is the 2D effective binding affinity, koff is the off-rate, mr and ml are the respective receptor and ligand densities measured in step 4, and Ac is the contact area. The curve-fit has two parameters, AcKa and koff, as Ac and Ka are lumped together and called collectively as effective 2D affinity. Its product with the off-rate is the effective 2D on-rate: Ackon = AcKa x koff. 
 The specific adhesion frequency Pa is calculated by subtraction of the nonspecific adhesion fraction (Pnonspecific) from the total measured adhesion (Pmeasured)1,21: 
 <img alt="Equation 2" src="/files/ftp_upload/3519/3519eq2.jpg" /></li>
</ol>
8. Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/3519/3519fig1.jpg" /> 
Figure 1 Determination of integrin &alpha;L&beta;2 site density on neutrophils. Neutrophils were first incubated with 1&mu;g/ml of E-selectin-Ig for 10min to match the experimental condition used in Figures 3,4 or without E-selectin-Ig, and then with saturating concentrations (10&mu;g/ml) of PE-conjugated anti-human CD11a mAb (Clone HI111, see Table of specific reagents and equipment) or irrelevant mouse IgG1 for control, washed, and analyzed immediately. Samples were read on BD LSR flow cytometer with standard QuantiBRITE PE calibration beads. Panel A shows fluorescence histograms of calibration beads (pink) together with those of E-selectin-Ig treated (blue color) or untreated cells (green color). Specific CD11a mAb staining is shown in solid curves and irrelevant isotype-matched control antibody staining is shown in dotted curves. Cells treated with E-selectin-Ig (presence in all washing steps and in FACS buffer) did not affect the CD11a density as seen from the comparison with untreated cells. Panel B shows the process of density quantification. Log10 was calculated for the mean fluorescent intensity (FI) of each peak value of four calibration bead histograms from Panel A (pink circles) and for the lot-specific PE molecules per bead (from the manufacturer). A linear regression of Log10 PE molecules per bead against Log10 fluorescence was plotted. For E-selectin treated cells the Log10 FI (y) values equal 3.99 (blue solid circle) and 2.23 (blue open circle) for specific mAb and control antibody, respectivly. We solved the linear equation for x (values are plotted as green and blue circles on Panel B). x = Log10 PE/cell and, as PE:mAb ratio was 1:1, the total number of &alpha;L&beta;2 on neutrophils was calculated as 9587. Surface density was calculated to be 43 molecules/&mu;m2, using 8.4&mu;m as the neutrophil diameter22. Density of ICAM-1 was similarly measured by flow cytometery using PE-anti-human CD54 mAb, which equaled 65 mol/&mu;m2.
<img alt="Figure 2" src="/files/ftp_upload/3519/3519fig2.jpg" /> 
Figure 2 Micropipette system schematics. Our micropipette system was assembled in house and consists of three subsystems: an imaging subsystem to allow one to observe, record and analyze movements of the micropipette-aspirated cell; a micromanipulation subsystem to enable one to select the cells from the cell chamber, and a pressure subsystem to allow one to aspirate the cells into micropipettes. The central piece of the imaging subsystem is inverted microscope (Olympus IMT-2 IMT2) with a 100x oil immersion 1.25 N.A. objective. The image is sent to a video cassette recorder through a charge couple device (CCD) camera. A video timer is coupled to the system to keep track of time. Each micropipette can be manipulated by a mechanical drive mounted on the microscope and finely positioned with a three-axis hydraulic micromanipulator. Mechanical manipulators from Newport could be used as well. One of the micropipette holders is mounted on a piezoelectric translator, the driver of which is controlled by a computer LabView code (available upon request) and the signal translates through a DAQ board via a voltage amplifier (homemade) to the piezo actuator. This allows one to move the pipette precisely and repeatably in an adhesion test cycle. The pressure regulation subsystem is used to control suction during the experiment. A hydraulic line connects the micropipette holder to a fluid reservoir. A fine mechanical positioner allows the height of the reservoir to be precisely manipulated.
Micropipettes are generated using KIMAX melting point borosilicate glass capillary tubes (with outside diameter of 1.0&plusmn;0.07 mm and an inside diameter of 0.7&plusmn;0.07 mm. First, the micropipettes from capillary tubes are pulled using PN-30 Narishige' Magnetic Glass Microelectrode Horizontal Puller (Sutter Instruments Micropipette Puller is another puller option). Second, Microforge system (built in house) is used to cut the micropipettes to desired size opening. Commercial models of Microforge systems are available as well.
To avoid vibration of the micropipettes during the experiment, the microscope, along with the micromanipulators, is placed on an air suspension table.
<img alt="Figure 3" src="/files/ftp_upload/3519/3519fig3.jpg" /> 
Figure 3 Running adhesion frequency Fi for specific (A) and nonspecific (B) binding at 1s (red) and 10s (blue) contact times measured from repeated adhesion test cycles between RBCs coated with ICAM-1 (A) or hIgG (B) with human neutrophils expressing integrin &alpha;L&beta;2. Fi = (X1 + X2 +&hellip;+ Xi)/i (1 &le;i &le; n), where i is the test cycle index, Xi equals "1" (adhesion) or "0" (no adhesion). Fn (n=50) was used as the best estimate for adhesion probability.
<img alt="Figure 4" src="/files/ftp_upload/3519/3519fig4.jpg" /> 
Figure 4 Kinetics of ICAM-1 binding to neutrophil integrin &alpha;L&beta;2 (<img alt="Square" src="/files/ftp_upload/3519/3519sq1.jpg" />). Adhesion probability measured as shown in Figure 3 for three cell pairs at each contact time is averaged and plotted versus contact time. The chamber medium was HBSS with 1mM each of Ca2+ and Mg2+ plus 1&mu;g/ml of dimeric E-selectin-Ig to upregulate &alpha;L&beta;2 binding. To capture ICAM-1-Ig on RBCs, an intermediate step was added to incubate RBCs with 10&mu;g/ml capture antibody (biotinylated goat-anti-human Fc antibody, eBioscience) after the streptavidin incubation step. To control for nonspecific binding two different conditions were used: 1) RBCs coated with the anti-human-Fc capture antibody and incubated with human IgG instead of ICAM-1-Ig (O) and 2) neutrophils binding to RBCs not coated with the capture antibody (&Delta;). 10&mu;g/ml human IgG was added to the medium to minimize binding of E-selectin-Ig in solution to the capture antibody on the RBC surface. Nonspecific binding recorded as human IgG control curve was used to obtain a specific adhesion probability curve (<img alt="Square" src="/files/ftp_upload/3519/3519sq2.jpg" />) using Eq. 2. Fitting specific adhesion probability curve with Eq. 1 (solid line) returned effective binding affinity AcKa = 1.4&bull;10-4 &mu;m4 and koff = 0.3 s-1.
<table border="1">
 <tbody>
 <tr>
 <th>
 Reagent</th>
 <th>
 MW (g/mol)</th>
 <th>
 Concentration (mM)</th>
 <th>
 Amount (g)</th>
 </tr>
 <tr>
 <td>Adenine</td>
 <td>135.13</td>
 <td>2</td>
 <td>0.27</td>
 </tr>
 <tr>
 <td>D-glucose (dextrose)</td>
 <td>180.16</td>
 <td>110</td>
 <td>19.82</td>
 </tr>
 <tr>
 <td>D-Mannitol</td>
 <td>182.17</td>
 <td>55</td>
 <td>10.02</td>
 </tr>
 <tr>
 <td>Sodium Chloride (NaCl)</td>
 <td>58.44</td>
 <td>50</td>
 <td>2.92</td>
 </tr>
 <tr>
 <td>Sodium Phosphate, Dibasic (Na HPO )</td>
 <td>141.95</td>
 <td>20</td>
 <td>2.84</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>146.15</td>
 <td>10</td>
 <td>1.46</td>
 </tr>
 </tbody>
</table>
Table 1. EAS-45 buffer preparation (1L).
<table border="1">
 <tbody>
 <tr>
 <td>25 mg biotin-XHS in 550 &mu;l of DMF</td>
 <td>0.1M biotin solution</td>
 </tr>
 <tr>
 <td>1:10 dilution of 0.1 biotin w/DMF</td>
 <td>0.01M biotin solution</td>
 </tr>
 <tr>
 <td>1:100 dilution of 0.1 biotin w/ DMF</td>
 <td>0.001M biotin solution</td>
 </tr>
 </tbody>
</table>
Table 2. Preparation of the biotin solution.
<table border="1">
 <tbody>
 <tr>
 <th>
 biotin final concentration (&mu;M)</th>
 <th>
 4</th>
 <th>
 10</th>
 <th>
 20</th>
 <th>
 50</th>
 <th>
 100</th>
 <th>
 160</th>
 </tr>
 <tr>
 <td>RBCs pellet stock (&mu;l)</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 </tr>
 <tr>
 <td>1x PBS (&mu;l)</td>
 <td>179.2</td>
 <td>178</td>
 <td>176</td>
 <td>179</td>
 <td>178</td>
 <td>176.8</td>
 </tr>
 <tr>
 <td>0.1M borate buffer (&mu;l)</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 <td>10</td>
 </tr>
 <tr>
 <td>0.01M biotin solution (&mu;l)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1</td>
 <td>2</td>
 <td>3.2</td>
 </tr>
 <tr>
 <td>0.001M biotin solution (&mu;l)</td>
 <td>0.8</td>
 <td>2</td>
 <td>4</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 3. Biotinylation of RBCs.

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Physiol. 69, 1767-1778 (1990).</li><li>Li, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+and+kinetic+analysis+of+Fcgamma+receptor+IIIa+(CD16a)+binding+to+IgG+ligands.">Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands.</a> J. Biol. Chem. 282, 6210-6221 (2007).</li><li>Chen, W., Zarnitsyna, V. I., Sarangapani, K. K., Huang, J., 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+Receptor-Ligand+Binding+Kinetics+on+Cell+Surfaces:+From+Adhesion+Frequency+to+Thermal+Fluctuation+Methods.">Measuring Receptor-Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods.</a> Cell. Mol. Bioeng. 1, 276-288 (2008).</li><li>Thoumine, O., Kocian, P., Kottelat, A., Meister, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+binding+of+fibroblasts+to+fibronectin:+optical+tweezers+experiments+and+probabilistic+analysis.">Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis.</a> Eur. Biophys. J. 29, 398-408 (2000).</li><li>Ounkomol, C., Xie, H., Dayton, P. A., Heinrich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+horizontal+force+probe+for+mechanical+tests+on+pipette-held+cells,+particles,+and+membrane+capsules.">Versatile horizontal force probe for mechanical tests on pipette-held cells, particles, and membrane capsules.</a> Biophys. J. 96, 1218-1231 (2009).</li><li>Piper, J. W., Swerlick, R. 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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Memory+in+receptor-ligand-mediated+cell+adhesion.">Memory in receptor-ligand-mediated cell adhesion.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 18037-18042 (2007).</li></ol>

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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Bioengineering

14.9K Views.  Georgia Institute of Technology. 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 αLβ2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

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

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to&#160;glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly&#160;to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to &#62;10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

The goal of this protocol is to demonstrate how to prepare serial crystallography samples for data collection on a high viscous injector, Lipidico, recently commissioned at the Australian synchrotron.

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...