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

Summary

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.

Abstract

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 Fcγ receptors with IgG Fc^1-6, selectins with glycoconjugate ligands^6-9, integrins with ligands^10-13, homotypical cadherin binding¹⁴, T cell receptor and coreceptor with peptide-major histocompatibility complexes^15-19.

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 molecules^15,17,19.

The method has also been used to study the concurrent binding of dual receptor-ligand species^3,4, 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 α_Lβ₂ on neutrophils with dimeric E-selectin in the solution to activate α_Lβ₂.

Protocol

1. RBCs isolation from the whole blood

1. 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°C for storage.

Note: Step 1.2 should be performed by a trained medical professional such as a nurse, with an Institutional Review Board approved protocol.

2. 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.
3. 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°C. Repeat twice.
4. Add 10ml cold and sterile PBS, centrifuge for 5min @ 1000rpm, 4°C. Remove the supernatant. Repeat 4 times (Total of 5 times).
5. Wash with sterile EAS-45 (5min @ 1000rpm, 4°C) twice. During last washing move RBCs into a new sterile 15ml tube.
6. Resuspend RBCs in10ml EAS-45.

2. RBCs biotinylation

1. Take the tube with RBCs from step 1. Remove all supernatant after centrifuging for 5min @ 1000rpm, 4°C. Put 10μl 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).
2. Make fresh Biotin-X-NHS/DMF 1:10, 1:100 dilutions according to Table II.
3. Add 10μl of 0.1M borate buffer to each vial.
4. Add calculated amount of biotin solution to each vial (see Table II as example) and vortex immediately.
5. Place each RBC vial inside a 50ml conical tube and incubate on rotator for 30min at room temperature.
6. Wash each vial 3 times with 800μl of EAS-45 for 2min @ 2000rpm.
7. Add 100μl of EAS-45 to each vial and store at 4°C. In each 1μl of the final solution now should be ˜1mln of RBCs.

3. Functionalizing the biotin-linked ligands* on RBCs

1. Prepare 2mg/ml streptavidin solution according to manufacturer's instructions. Aliqouted solution may be stored at -20°C.
2. Mix an equal amount of RBCs from step 2.7 (10μl) with streptavidin solution, vortex immediately and incubate on rotator for 30min at 4°C.
3. Wash 3 times with 500μl of EAS-45 for 2min @ 2000rpm. Add 15μl EAS-45/ 1% BSA for storage.
4. Mix equal amount of RBCs from step 3.3 (10μl) with 20μg/ml ligand solution, vortex immediately and incubate on rotator for 30min at room temperature.
5. Wash two times with 500μl of EAS-45/ 1% BSA for 2min @ 2000rpm. Add 15μl of EAS-45/1% BSA for storage.

*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

1. 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.
2. Analyze samples prepared in step 4.1 by flow cytometery with corresponding fluorescent calibration beads. Calculate the densities as shown in Fig. 1.

5. Preparation for micropipette and cell chamber

1. Pull micropipettes from capillary tubes using PN-30 Narishige' Magnetic Glass Microelectrode Horizontal Puller or Sutter Instruments Micropipette Puller.
2. Use micromanipulator with microforge to adjust the tip of the pulled micropipette to a desired size (usually the required diameter ranges from 1-5μm depending on the size of the cells to be used in the study).
3. 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.
4. Add the receptor- and ligand-bearing cells to the chamber.

6. Micropipette adhesion frequency assay

1. 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.
2. 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.
3. 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.
4. 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.

7. Data analysis

1. Fit the specific adhesion frequency P_a 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:
   
   where K_a is the 2D effective binding affinity, k_off is the off-rate, m_r and m_l are the respective receptor and ligand densities measured in step 4, and A_c is the contact area. The curve-fit has two parameters, A_cK_a and k_off, as A_c and K_a are lumped together and called collectively as effective 2D affinity. Its product with the off-rate is the effective 2D on-rate: A_ck_on = A_cK_a x k_off.
   The specific adhesion frequency P_a is calculated by subtraction of the nonspecific adhesion fraction (P_nonspecific) from the total measured adhesion (P_measured)^1,21:
   

8. Representative Results:

Figure 1 Determination of integrin α_Lβ₂ site density on neutrophils. Neutrophils were first incubated with 1μg/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 (10μg/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 α_Lβ₂ on neutrophils was calculated as 9587. Surface density was calculated to be 43 molecules/μm², using 8.4μm as the neutrophil diameter²². Density of ICAM-1 was similarly measured by flow cytometery using PE-anti-human CD54 mAb, which equaled 65 mol/μm².

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±0.07 mm and an inside diameter of 0.7±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.

Figure 3 Running adhesion frequency F_i 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 α_Lβ₂. F_i = (X₁ + X₂ +…+ X_i)/i (1 ≤i ≤ n), where i is the test cycle index, X_i equals "1" (adhesion) or "0" (no adhesion). F_n (n=50) was used as the best estimate for adhesion probability.

Figure 4 Kinetics of ICAM-1 binding to neutrophil integrin α_Lβ₂ (). 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 1μg/ml of dimeric E-selectin-Ig to upregulate α_Lβ₂ binding. To capture ICAM-1-Ig on RBCs, an intermediate step was added to incubate RBCs with 10μg/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 (Δ). 10μg/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 () using Eq. 2. Fitting specific adhesion probability curve with Eq. 1 (solid line) returned effective binding affinity A_cK_a = 1.4•10^-4 μm4 and k_off = 0.3 s^-1.

Reagent	MW (g/mol)	Concentration (mM)	Amount (g)
Adenine	135.13	2	0.27
D-glucose (dextrose)	180.16	110	19.82
D-Mannitol	182.17	55	10.02
Sodium Chloride (NaCl)	58.44	50	2.92
Sodium Phosphate, Dibasic (Na HPO )	141.95	20	2.84
L-glutamine	146.15	10	1.46

Table 1. EAS-45 buffer preparation (1L).

25 mg biotin-XHS in 550 μl of DMF	0.1M biotin solution
1:10 dilution of 0.1 biotin w/DMF	0.01M biotin solution
1:100 dilution of 0.1 biotin w/ DMF	0.001M biotin solution

Table 2. Preparation of the biotin solution.

biotin final concentration (μM)	4	10	20	50	100	160
RBCs pellet stock (μl)	10	10	10	10	10	10
1x PBS (μl)	179.2	178	176	179	178	176.8
0.1M borate buffer (μl)	10	10	10	10	10	10
0.01M biotin solution (μl)				1	2	3.2
0.001M biotin solution (μl)	0.8	2	4

Table 3. Biotinylation of RBCs.

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x PBS	BioWhittaker	17-517Q	Dilute to 1x with deionized water prior to use
Vacutainer EDTA	BD Biosciences	366643	RBCs isolation
10ML PK100
Histopaque 1077	Sigma-Aldrich	10771	RBCs isolation
Adenine	Sigma-Aldrich	A2786	EAS-45 preparation
D-glucose (dextrose)	Sigma-Aldrich	G7528	EAS-45 preparation
D-Mannitol	Sigma-Aldrich	6360	EAS-45 preparation
Sodium Chloride (NaCl)	Sigma-Aldrich	S7653	EAS-45 preparation
Sodium Phosphate, Dibasic (Na₂HPO₄)	Fisher Scientific	S374	EAS-45 preparation
L-glutamine	Sigma-Aldrich	G5763	EAS-45 preparation
Biotin-X-NHS	Calbiochem	203188	RBCs biotinylation
Dimethylformamide (DMF)	Thermo Fisher Scientific, Inc.	20673	RBCs biotinylation
Borate Buffer (0.1M)	Electron Microscopy Sciences	11455-90	RBCs biotinylation
Streptavidin	Thermo Fisher Scientific, Inc.	21125	Ligand functionalizing
BSA	Sigma-Aldrich	A0336	Ligand functionalizing
Quantibrite PE Beads	BD Biosciences	340495	Density quantification
Flow cytometer	BD Biosciences	BD LSR II	Density quantification
Capillary Tube 0.7-1.0mm x 30"	Kimble Chase	46485-1	Micropipette pulling
Mineral Oil	Fisher Scientific	BP2629-1	Chamber assembly
Microscope Cover Glass	Fisher Scientific	12-544-G	Chamber assembly
PE α-human CD11a Clone HI 111	eBioscience	12-0119-71	Reagent for Fig.1
PE anti-human CD54	eBioscience	12-0549	Reagent for Fig.1
Mouse IgG1 Isotype Control PE	eBioscience	12-4714	Reagent for Fig.1
hydraulic micromanipulator	Narishige International	MO-303	Micropipette system
Mechanical manipulator	Newport Corp.	461-xyz-m, SM-13, DM-13	Micropipette system
piez–lectric translator	Physik Instruments	P-840	Micropipette system
LabVIEW	National Instruments	Version 8.6	Micropipette system
DAQ board	National Instruments	USB-6008	Micropipette system
Optical table	Kinetic Systems	5200 Series	Micropipette system