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....

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