title: Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization.
abstract: 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.
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm-Meyers Allé 4, 8000 Aarhus C, Denmark; Department of Molecular Biology and Genetics, Aarhus University, C.F. Moellers Allé 3, 8000 Aarhus C, Denmark. Electronic address: sofieselmer@immunology.au.dk.
authors.forename: Sofie Selmer
authors.initials: SS
authors.lastname: Andersen
authors.forename: Malene
authors.initials: M
authors.lastname: Hvid
authors.forename: Finn Skou
authors.initials: FS
authors.lastname: Pedersen
authors.forename: Bent
authors.initials: B
authors.lastname: Deleuran
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000276:immunology*; D053699:Interleukin-7 Receptor alpha Subunit / Q000276:immunology*; D008856:Microscopy, Fluorescence / Q000379:methods*; D011485:Protein Binding; D055503:Protein Multimerization; D013601:T-Lymphocytes / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D053631:Interleukin Receptor Common gamma Subunit; D053699:Interleukin-7 Receptor alpha Subunit
keywords: 4′,6-diamidino-2-phenylindole; APC; CFSE; Cytokine receptor dimerization; DAPI; EGFR; Flow cytometry; IL; IL-7Rα; MFI; PE; PLA; Proximity ligation assay; RCA; TSLP; allophycocyanin; carboxy-fluorescein diacetate succinimidyl ester; common gamma chain; epidermal growth factor receptor; interleukin; interleukin 7 receptor alpha; median fluorescence intensity; phycoerythrin; proximity ligation assay; rolling circle amplification; thymic stromal lymphopoietin; γ(c)