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Updated: Jun 19, 2026
Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery
Published on: October 4, 2018
Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets
P Lanuti1, S Fuhrmann, R Lachmann
1Division of Medicine, Brighton and Sussex Medical School, Brighton, United Kingdom.
Insights
This study presents a novel multi-parameter flow cytometry protocol for detailed T cell analysis. The method simultaneously measures surface markers, intracellular signaling, and DNA synthesis for comprehensive immune cell profiling.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Multi-color flow cytometry is crucial for analyzing complex immune cell populations at single-cell resolution.
- T cell compartment analysis demands simultaneous measurement of multiple markers for lineage, phenotype, and function.
- Existing methods limit the simultaneous assessment of intracellular signaling, proliferation, and DNA synthesis.
Purpose of the Study:
- To develop and optimize a comprehensive flow cytometry protocol for simultaneous analysis of T cell lineage, phenotype, function, and intracellular signaling.
- To enable the simultaneous acquisition of data on surface markers, intracellular cytokines, kinase phosphorylation, cell proliferation, and DNA content.
- To provide mechanistic and kinetic insights into subset-specific T cell signaling pathways.
Main Methods:
- Optimization of antibody/fluorochrome combinations, permeabilization, fixation, and labeling times for improved DNA staining.
- Development of a multi-parameter flow cytometry protocol integrating surface marker expression, intracellular cytokine production, kinase phosphorylation status, cell proliferation, and DNA content.
- Application of the optimized protocol to T cells responding to specific antigens.
Main Results:
- A robust protocol was established allowing simultaneous measurement of diverse T cell characteristics.
- Optimized procedures enhanced DNA staining quality across different cell types.
- The method successfully revealed subset-specific signaling related to cytokine production and DNA synthesis in antigen-stimulated T cells.
Conclusions:
- The developed flow cytometry protocol offers an unprecedented capacity for simultaneous, high-resolution analysis of T cell responses.
- This comprehensive approach provides valuable mechanistic and kinetic information previously unattainable.
- The protocol is applicable to studying complex T cell signaling and function in various immunological contexts.
Abstract:
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.
