title: Microfluidic Proximity Ligation Assay for Profiling Signaling Networks with Single-Cell Resolution.
abstract: 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.
authors.affiliation: Microfluidic and Biological Engineering, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110, Freiburg, Germany.|BIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79108, Freiburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Blazek
authors.affiliation: BIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79108, Freiburg, Germany.|Center for Biological Systems Analysis (ZBSA), University of Freiburg, 79104, Freiburg, Germany.
authors.forename: Günter
authors.initials: G
authors.lastname: Roth
authors.affiliation: BIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79108, Freiburg, Germany.|Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110, Freiburg, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Zengerle
authors.affiliation: Microfluidic and Biological Engineering, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110, Freiburg, Germany. Matthias.meier@imtek.uni-freiburg.de.|BIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79108, Freiburg, Germany. Matthias.meier@imtek.uni-freiburg.de.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meier
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000295:instrumentation / Q000379:methods*
chemical_list: D010982:Platelet-Derived Growth Factor; D051057:Proto-Oncogene Proteins c-akt
keywords: Cell culture; Cell signaling; Microfluidics; Polydimethylsiloxane; Proximity ligation assay; Single-cell segmentation