Aarhus University Seal / Aarhus Universitets segl

Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Many cytokine receptors are cell surface proteins that promiscuously combine to form active signalling homo- or heterodimers. Thus, receptor chain dimerization can be viewed as a direct measure of a high probability of intracellular signalling by specific cytokines. Proximity ligation assay (PLA) is an antibody-based method for selective and highly sensitive detection of protein interactions by microscopy. As proof of concept, the aim of this study was to combine antibodies towards interleukin 7 receptor alpha (IL-7Rα) and the common gamma chain (γc) with PLA and flow cytometry to enable the detection of IL-7 receptor heterodimers. The presence of IL-7 receptor heterodimers on the surface of the HPB-ALL T cell line was detected by PLA and microscopy with a resolution of one complex per cell. Optimisation of the PLA reaction on cell suspensions identified buffer effects with critical importance for the flow cytometric outcome. In addition, blocking, fixation and incubation conditions were optimised to prevent unspecific antibody binding. PLA combined with flow cytometry very sensitively detected receptor heterodimers on the cell surface. Thus, the method is a powerful tool for the investigation of cytokine receptor dimerization.
Original languageEnglish
JournalCytokine
Volume64
Issue1
Pages (from-to)54-57
Number of pages4
ISSN1043-4666
DOIs
Publication statusPublished - Oct 2013

    Research areas

  • Proximity ligation assay, Flow cytometry, Cytokine receptor dimerization

See relations at Aarhus University Citationformats

ID: 56479509