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

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Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization. / Andersen, Sofie Selmer; Hvid, Malene; Pedersen, Finn Skou; Deleuran, Bent.

I: Cytokine, Bind 64, Nr. 1, 10.2013, s. 54-57.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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@article{86a3381da15848b1bc1847e742c0a7c5,
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.",
keywords = "Proximity ligation assay, Flow cytometry, Cytokine receptor dimerization",
author = "Andersen, {Sofie Selmer} and Malene Hvid and Pedersen, {Finn Skou} and Bent Deleuran",
note = "Copyright {\textcopyright} 2013 Elsevier Ltd. All rights reserved.",
year = "2013",
month = oct,
doi = "10.1016/j.cyto.2013.04.026",
language = "English",
volume = "64",
pages = "54--57",
journal = "Cytokine",
issn = "1043-4666",
publisher = "Academic Press",
number = "1",

}

RIS

TY - JOUR

T1 - Proximity ligation assay combined with flow cytometry is a powerful tool for the detection of cytokine receptor dimerization

AU - Andersen, Sofie Selmer

AU - Hvid, Malene

AU - Pedersen, Finn Skou

AU - Deleuran, Bent

N1 - Copyright © 2013 Elsevier Ltd. All rights reserved.

PY - 2013/10

Y1 - 2013/10

N2 - 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.

AB - 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.

KW - Proximity ligation assay

KW - Flow cytometry

KW - Cytokine receptor dimerization

U2 - 10.1016/j.cyto.2013.04.026

DO - 10.1016/j.cyto.2013.04.026

M3 - Journal article

C2 - 23726671

VL - 64

SP - 54

EP - 57

JO - Cytokine

JF - Cytokine

SN - 1043-4666

IS - 1

ER -