Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets

Lanuti, P, Fuhrmann, S, Lachmann, R, Marchisio, M, Miscia, S and Kern, F (2009) Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets. International Journal of Immunopathology and Pharmacology, 22 (3). pp. 689-698. ISSN 0394-6320

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Multi-colour flow cytometry is the only technological platform that can analyse the highly complex cellular composition of the immune system in parallel and at a single cell resolution. Analysis of the T cell compartment, in particular, requires the simultaneous measurement of multiple markers in order to account for lineage, phenotype and function. Flow cytometry also enables the analysis of intracellular signalling events. By combining the expression of surface markers, intracellular cytokines, phosphorylated versus unphosphorylated kinases, cell proliferation and DNA profile, mechanistic and kinetic information of subset-specific signalling may be obtained: this has not previously been achieved. Here we present a protocol which permits all of these aspects to be explored simultaneously. By comparing basic procedures previously described we were able to optimise different variables, including the choice of antibody/fluorochrome pairs, permeabilisation, fixation and labelling time, to obtain the best DNA staining of different cell types. We applied this method to study subset-specific signalling related to cytokine production and DNA synthesis in T cells responding to specific antigens.

Item Type: Article
Keywords: Biological Markers/metabolism Brefeldin A/pharmacology *Cell Cycle/drug effects Cell Cycle Proteins/*metabolism Cells, Cultured Cytokines/*metabolism DNA Replication Enterotoxins/pharmacology Female *Flow Cytometry Humans Immunophenotyping/*methods Kinetics *Lymphocyte Activation/drug effects Male Phosphoproteins/*metabolism Signal Transduction Staining and Labeling T-Lymphocyte Subsets/drug effects/immunology/*metabolism
Schools and Departments: Brighton and Sussex Medical School > Clinical and Experimental Medicine
Subjects: Q Science > QR Microbiology > QR0180 Immunology
Depositing User: Florian Kern
Date Deposited: 25 Oct 2012 13:15
Last Modified: 25 Oct 2012 13:15
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