High-Content Imaging for Large-Scale Detection of Low-AffinityExtracellular Protein Interactions

摘要

Extracellular protein interactions coordinate cellular responses with their localenvironment and have important roles in pathogen invasion and disease. Due totechnical challenges associated with studying binding events at the cellsurface, the systematic and reliable identification of novel ligand–receptorpairs remains difficult. Here, we describe the development of a cell-based assayusing large-scale transient transfections and high-content imaging (HCI) todetect extracellular binding events. We optimized the parameters for efficienttransfection of human cells with cDNA plasmids encoding full-length cell surfacereceptors in 384-well plates. Using a range of well-characterized structurallydiverse low-affinity cell surface interactions, we show that transfected cellsprobed with highly avid ligands can be used to successfully identifyligand–receptor pairs using an HCI platform and automated image analysissoftware. To establish the high-throughput potential of this approach, we alsoscreened a pool of ligands against a collection of 2455 cell surface expressionclones and found that known ligand–receptor interactions could be robustly andconsistently detected across the library using this technology.