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Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases

Flow cytometric analysis of p38 mitogen-activated protein kinase (p38 MAPK) signaling cascade is optimally achieved by methanol permeabilization protocols. Such protocols suffer from the difficulties to accurately detect intracellular cytokines and surface epitopes of infrequent cell subpopulations,...

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Autores principales: Mavropoulos, Athanasios, Bogdanos, Dimitrios P., Liaskos, Christos, Orfanidou, Timoklia, Simopoulou, Theodora, Zafiriou, Efterpi, Sakkas, Lazaros I., Rigopoulou, Eirini I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987974/
https://www.ncbi.nlm.nih.gov/pubmed/24741615
http://dx.doi.org/10.1155/2014/671431
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author Mavropoulos, Athanasios
Bogdanos, Dimitrios P.
Liaskos, Christos
Orfanidou, Timoklia
Simopoulou, Theodora
Zafiriou, Efterpi
Sakkas, Lazaros I.
Rigopoulou, Eirini I.
author_facet Mavropoulos, Athanasios
Bogdanos, Dimitrios P.
Liaskos, Christos
Orfanidou, Timoklia
Simopoulou, Theodora
Zafiriou, Efterpi
Sakkas, Lazaros I.
Rigopoulou, Eirini I.
author_sort Mavropoulos, Athanasios
collection PubMed
description Flow cytometric analysis of p38 mitogen-activated protein kinase (p38 MAPK) signaling cascade is optimally achieved by methanol permeabilization protocols. Such protocols suffer from the difficulties to accurately detect intracellular cytokines and surface epitopes of infrequent cell subpopulations, which are removed by methanol. To overcome these limitations, we have modified methanol-based phosphoflow protocols using several commercially available antibody clones suitable for surface antigens, intracellular cytokines, and p38 MAPK. These included markers of B cells (CD19, CD20, and CD22), T cells (CD3, CD4, and CD8), NK (CD56 and CD7), and dendritic cells (CD11c). We have also tested surface markers of costimulatory molecules, such as CD27. We have successfully determined simultaneous expression of IFN-γ, as well as IL-10, and phosphorylated p38 in cell subsets. The optimized phosphoflow protocol has also been successfully applied in peripheral blood mononuclear cells or purified cell subpopulations from patients with various autoimmune diseases. In conclusion, our refined phosphoflow cytometric approach allows simultaneous detection of p38 MAPK activity and intracellular cytokine expression and could be used as an important tool to study signaling cascades in autoimmunity.
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spelling pubmed-39879742014-04-16 Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases Mavropoulos, Athanasios Bogdanos, Dimitrios P. Liaskos, Christos Orfanidou, Timoklia Simopoulou, Theodora Zafiriou, Efterpi Sakkas, Lazaros I. Rigopoulou, Eirini I. J Immunol Res Research Article Flow cytometric analysis of p38 mitogen-activated protein kinase (p38 MAPK) signaling cascade is optimally achieved by methanol permeabilization protocols. Such protocols suffer from the difficulties to accurately detect intracellular cytokines and surface epitopes of infrequent cell subpopulations, which are removed by methanol. To overcome these limitations, we have modified methanol-based phosphoflow protocols using several commercially available antibody clones suitable for surface antigens, intracellular cytokines, and p38 MAPK. These included markers of B cells (CD19, CD20, and CD22), T cells (CD3, CD4, and CD8), NK (CD56 and CD7), and dendritic cells (CD11c). We have also tested surface markers of costimulatory molecules, such as CD27. We have successfully determined simultaneous expression of IFN-γ, as well as IL-10, and phosphorylated p38 in cell subsets. The optimized phosphoflow protocol has also been successfully applied in peripheral blood mononuclear cells or purified cell subpopulations from patients with various autoimmune diseases. In conclusion, our refined phosphoflow cytometric approach allows simultaneous detection of p38 MAPK activity and intracellular cytokine expression and could be used as an important tool to study signaling cascades in autoimmunity. Hindawi Publishing Corporation 2014 2014-02-03 /pmc/articles/PMC3987974/ /pubmed/24741615 http://dx.doi.org/10.1155/2014/671431 Text en Copyright © 2014 Athanasios Mavropoulos et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Mavropoulos, Athanasios
Bogdanos, Dimitrios P.
Liaskos, Christos
Orfanidou, Timoklia
Simopoulou, Theodora
Zafiriou, Efterpi
Sakkas, Lazaros I.
Rigopoulou, Eirini I.
Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases
title Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases
title_full Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases
title_fullStr Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases
title_full_unstemmed Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases
title_short Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases
title_sort flow cytometric detection of p38 mapk phosphorylation and intracellular cytokine expression in peripheral blood subpopulations from patients with autoimmune rheumatic diseases
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987974/
https://www.ncbi.nlm.nih.gov/pubmed/24741615
http://dx.doi.org/10.1155/2014/671431
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