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Open Access Green as soon as Postprint is submitted to ZB.
Microfluidic proximity ligation assay for profiling signaling networks with single-cell resolution.
Methods Mol. Biol. 1346, 169-184 (2015)
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.
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Publication type
Article: Journal article
Document type
Scientific Article
Keywords
Cell Culture ; Cell Signaling ; Microfluidics ; Polydimethylsiloxane ; Proximity Ligation Assay ; Single-cell Segmentation
ISSN (print) / ISBN
1064-3745
e-ISSN
1940-6029
Journal
Methods in Molecular Biology
Quellenangaben
Volume: 1346,
Pages: 169-184
Publisher
Springer
Publishing Place
Berlin [u.a.]
Non-patent literature
Publications
Reviewing status
Peer reviewed
Institute(s)
Helmholtz Pioneer Campus (HPC)