title: An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells.
abstract: Phosphoflow cytometry is increasingly being used as a tool for the discovery of biomarkers used in the treatment and monitoring of disease and therapy. The ability to measure numerous phospho-protein targets simultaneously at a single cell level accurately and rapidly provides significant advantages over other methods. We here discuss important considerations required to successfully implement these methods. Three different blood collection tubes (lithium-heparin tubes, CPT with sodium citrate and CPT with sodium heparin) were evaluated, with PBMC isolated through lithium-heparin tubes/lymphoprep displaying reduced basal and increased stimulation induced phosphorylation compared to the other two methods. Further, we provide a protocol outlining an 8 color assay developed for the study of intracellular signaling in peripheral blood mononuclear cells. The assay allows for the quantitative measurement of the phospho-proteins ERK1/2, NF-κB p65, Stat1 (Y701), Stat1 (S727), Stat3 (Y705), Stat3 (S727), Stat4 (Y693), p38 and Stat5 (Y694), as well as the identification of T cells, B cells, natural killer cells and monocytes. The assay additionally incorporates fluorescent cell barcoding, reducing assay costs and increasing throughput while increasing data robustness. Inter-assay precision was assessed over a month long period for all experimental variables (phospho-protein measured, cell type and stimulant). Coefficient of variations (CVs) calculated from process triplicates of normalized median fluorescence intensity (MFI) of the phospho-proteins displayed median CVs under 10% when grouped according to cell type, stimulation agent and phospho-protein measured, while the CV for each triplicate did not exceed 20%.
authors.affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Jonas Lies vei 87, 5021 Bergen, Norway. Electronic address: Richard.Davies@uib.no.
authors.forename: Richard
authors.initials: R
authors.lastname: Davies
authors.affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Jonas Lies vei 87, 5021 Bergen, Norway.
authors.forename: Petra
authors.initials: P
authors.lastname: Vogelsang
authors.affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Jonas Lies vei 87, 5021 Bergen, Norway; Department of Rheumatology, Haukeland University Hospital, 5021 Bergen, Norway.
authors.forename: Roland
authors.initials: R
authors.lastname: Jonsson
authors.affiliation: Broegelmann Research Laboratory, Department of Clinical Science, University of Bergen, Jonas Lies vei 87, 5021 Bergen, Norway.
authors.forename: Silke
authors.initials: S
authors.lastname: Appel
mesh_terms: D001402:B-Lymphocytes / Q000166:cytology*; D018929:Cell Culture Techniques; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009000:Monocytes / Q000166:cytology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology*
chemical_list: D010750:Phosphoproteins
keywords: Barcoding; Flow cytometry; Multiparameter; PBMC; Phosphorylation; Signaling