Microfluidic Proximity Ligation Assay for Profiling Signaling Networks with Single-Cell Resolution

The proximity ligation assay (PLA) is a technique that can be used to characterize proteins, protein-protein interactions, and protein modifications at the single-cell level. Image-based in situ detection of proteins using PLA is a quantitative method with a high degree of sensitivity and specificity. The miniaturization and parallelization of the PLA onto a microfluidic chip and concurrent use of an automated cell-culture system increase the throughput of this technology. Here, we describe the performance of PLA on a microfluidic chip. We provide protocols for on-chip cell culture, time-shifted cell stimulation and fixation, PLA implementation, and computational image analysis in order to achieve single-cell resolution. As a proof of concept, we studied the phosphorylation of Akt in response to stimulation with platelet-derived growth factor.