Fluorescence-Based Measurements of Two-Dimensional Affinity in Membrane Interfaces

Binding between ligands and receptors across cell contacts influences a range of biological processes including the formation of the immune synapse. The dissociation constant (K_d = 1/affinity) of the interaction corresponds to the concentration of ligands where half of the receptors in the contact have bound a ligand. In this chapter, we outline how to measure this two-dimensional affinity using model cell membranes called supported lipid bilayers (SLBs) functionalized with fluorescently labeled ligands that bind to cells containing the corresponding receptor. The affinity is calculated from the accumulation of ligands at the cell-SLB interface, while the use of different fluorescent tags, and/or unlabeled molecules, makes it possible to include various binding pairs in the contact to better mimic the conditions of binding in vivo.