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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| Format: | Article |
| Language: | English |
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Wiley
2014-01-01
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| Series: | Journal of Immunology Research |
| Online Access: | http://dx.doi.org/10.1155/2014/671431 |
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| author | Athanasios Mavropoulos Dimitrios P. Bogdanos Christos Liaskos Timoklia Orfanidou Theodora Simopoulou Efterpi Zafiriou Lazaros I. Sakkas Eirini I. Rigopoulou |
| author_facet | Athanasios Mavropoulos Dimitrios P. Bogdanos Christos Liaskos Timoklia Orfanidou Theodora Simopoulou Efterpi Zafiriou Lazaros I. Sakkas Eirini I. Rigopoulou |
| author_sort | Athanasios Mavropoulos |
| collection | DOAJ |
| 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. |
| format | Article |
| id | doaj-art-469beefc4163485d89f03ebda73b442b |
| institution | Kabale University |
| issn | 2314-8861 2314-7156 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Journal of Immunology Research |
| spelling | doaj-art-469beefc4163485d89f03ebda73b442b2025-02-03T01:10:32ZengWileyJournal of Immunology Research2314-88612314-71562014-01-01201410.1155/2014/671431671431Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic DiseasesAthanasios Mavropoulos0Dimitrios P. Bogdanos1Christos Liaskos2Timoklia Orfanidou3Theodora Simopoulou4Efterpi Zafiriou5Lazaros I. Sakkas6Eirini I. Rigopoulou7Department of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, GreeceDepartment of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, GreeceDepartment of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, GreeceCellular Immunotherapy and Molecular Immunodiagnostics, Biomedical Section, Centre for Research & Technology Hellas (CE.R.T.H.)/Institute for Research and Technology-Thessaly (I.RE.TE.TH), 41222 Larissa, GreeceDepartment of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, GreeceDepartment of Dermatology, School of Health Sciences, Faculty of Medicine, University of Thessaly, Biopolis, 41110 Larissa, GreeceDepartment of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, GreeceDepartment of Internal Medicine, School of Health Sciences, Faculty of Medicine, University of Thessaly, Biopolis, 41110 Larissa, GreeceFlow 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.http://dx.doi.org/10.1155/2014/671431 |
| spellingShingle | Athanasios Mavropoulos Dimitrios P. Bogdanos Christos Liaskos Timoklia Orfanidou Theodora Simopoulou Efterpi Zafiriou Lazaros I. Sakkas Eirini I. Rigopoulou Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases Journal of Immunology Research |
| 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 |
| url | http://dx.doi.org/10.1155/2014/671431 |
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