A Flow Cytometry-Based Assay to Quantify the Binding of Transmembrane Ligands to Their Cognate Receptors Using...

The binding of transmembrane (TM) ligands to their cognate TM receptors on neighboring cells governs intercellular adhesion and direct cell-cell communication. However, these interactions are difficult to study in vitro because they depend on membrane presentation, ligand orientation, receptor clustering, and avidity, features often not captured by soluble recombinant ligands or cell-free assays. Here, we describe a flow cytometry-based assay using fluorescent, lentiviral virus-like particles (VLPs) displaying TM ligands to quantify binding to their receptors on target cells. Fluorescent VLPs are generated in-house by plasmid transfection in HEK293T cells and enable direct fluorescent detection without fluorochrome-conjugated secondary antibodies. The system is modular and readily accommodates engineered ligand constructs, including patient-derived variants. We applied this platform to generate ICAM-1-displaying fluorescent VLPs and to study human LFA-1 function in patient-derived leukocytes. This protocol provides a detailed workflow for VLP production and in vitro binding assays, offering a simple, quantitative, and cost-effective approach for studying TM ligand-receptor interactions in a membrane context. The system is well-suited for mechanistic studies, functional assessment of patient-derived variants, and direct binding assays using patient-derived cells. Integrating the assay into multicolor flow cytometry panels enables simultaneous immunophenotyping and quantification of up to four ligand-receptor interactions at single-cell resolution. Key features • Quantifies TM ligand-receptor binding in a membrane context using fluorescent VLPs and flow cytometry. • Fully in-house, modular system based on plasmid transfection in HEK293T cells, without reliance on recombinant ligands or fluorochrome-conjugated secondary antibodies. • Supports testing of engineered ligand variants, including patient-derived alleles, and direct functional studies on patient-derived cells. • Compatible with multicolor flow cytometry panels, enabling simultaneous immunophenotyping and quantification of up to four ligand-receptor interactions at single-cell resolution.