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Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets
journal contribution
posted on 2023-06-08, 12:41 authored by P Lanuti, S Fuhrmann, R Lachmann, M Marchisio, S Miscia, Florian KernFlorian KernMulti-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.
History
Publication status
- Published
Journal
International Journal of Immunopathology and PharmacologyISSN
0394-6320Publisher
University of Chieti -- Medical SchoolPublisher URL
Issue
3Volume
22Page range
689-698Department affiliated with
- Clinical and Experimental Medicine Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-10-25Usage metrics
Categories
No categories selectedKeywords
Biological Markers/metabolismBrefeldin A/pharmacology*Cell Cycle/drug effectsCell Cycle Proteins/*metabolismCellsCulturedCytokines/*metabolismDNA ReplicationEnterotoxins/pharmacologyFemale*Flow CytometryHumansImmunophenotyping/*methodsKinetics*Lymphocyte Activation/drug effectsMalePhosphoproteins/*metabolismSignal TransductionStaining and LabelingT-Lymphocyte Subsets/drug effects/immunology/*metabolism