title: Flow cytometric detection of p38 MAPK phosphorylation and intracellular cytokine expression in peripheral blood subpopulations from patients with autoimmune rheumatic diseases.
abstract: 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.
authors.affiliation: Department of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, Greece ; Cellular 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, Greece.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Mavropoulos
authors.affiliation: Department of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, Greece ; Cellular 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, Greece ; Division of Transplantation Immunology and Mucosal Biology, Institute of Liver Studies, King's College London School of Medicine King's College Hospital, London SE5 9RS, UK.
authors.forename: Dimitrios P
authors.initials: DP
authors.lastname: Bogdanos
authors.affiliation: Department of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, Greece ; Cellular 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, Greece.
authors.forename: Christos
authors.initials: C
authors.lastname: Liaskos
authors.affiliation: Cellular 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, Greece.
authors.forename: Timoklia
authors.identifier: 0000-0002-5485-2220
authors.initials: T
authors.lastname: Orfanidou
authors.affiliation: Department of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, Greece.
authors.forename: Theodora
authors.identifier: 0000-0003-2604-5090
authors.initials: T
authors.lastname: Simopoulou
authors.affiliation: Department of Dermatology, School of Health Sciences, Faculty of Medicine, University of Thessaly, Biopolis, 41110 Larissa, Greece.
authors.forename: Efterpi
authors.identifier: 0000-0002-6594-7292
authors.initials: E
authors.lastname: Zafiriou
authors.affiliation: Department of Rheumatology, Faculty of Medicine, School of Health Sciences, University of Thessaly, Biopolis, 41110 Larissa, Greece.
authors.forename: Lazaros I
authors.initials: LI
authors.lastname: Sakkas
authors.affiliation: Department of Internal Medicine, School of Health Sciences, Faculty of Medicine, University of Thessaly, Biopolis, 41110 Larissa, Greece.
authors.forename: Eirini I
authors.initials: EI
authors.lastname: Rigopoulou
mesh_terms: D000328:Adult; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D001327:Autoimmune Diseases / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D015415:Biomarkers; D003713:Dendritic Cells / Q000276:immunology / Q000473:pathology; D005260:Female; D015870:Gene Expression; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000235:genetics* / Q000276:immunology; D016753:Interleukin-10 / Q000235:genetics* / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology / Q000473:pathology; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D012216:Rheumatic Diseases / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D013601:T-Lymphocytes / Q000276:immunology / Q000473:pathology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000276:immunology
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; C508609:IL10 protein, human; D016753:Interleukin-10; D007371:Interferon-gamma; D048051:p38 Mitogen-Activated Protein Kinases
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