Fig. 1: Illustration of multi-well ephrin clustering assay.
From: Single-cell analysis of EphA clustering phenotypes to probe cancer cell heterogeneity
a Schematic representation of the experimental procedure. A silicon gasket comprising of nine chambers is placed on a clean coverslip. The coverslip is biofunctionalized with small unilamellar vesicles to form a supported lipid bilayer. The bilayer presents ephrinA1 ligands. Cells are seeded in each chamber, left for sedimentation, and incubation for an hour. The system is fixed and mounted on a coverslip for subsequent imaging. b, c Cartoons illustrating the EphA clustering assay. By sedimentation, cells contact the supported bilayer functionalized with fluorescent chimera of the ephrinA1 ligand. Upon receptor/ligand recognition (step 1), active receptors dimerize and phosphorylate (step 2), initiating the signaling cascade. In response to the initial signaling, active transport of receptors created signaling clusters. Their aggregation is imaged using ephrinA1 tagged with Alexa 568. d Examples of images of noninvasive breast cancer cells (left, MCF10A) and an (e) invasive breast cancer cells (right, MDA MB231) presenting different cluster morphologies (Scale bar: 5 µm). Scoring of individual cells based on quantification of the cluster morphologies is used as a proxy to define the heterogeneity of cellular states within a population.
