Analysing phosphorylation-based signalling networks by phospho flow cytometry

Analysis of signalling events by classical biochemical approaches is limited as the outcome is an averaged readout for protein activation of a single protein within a cell population. This is a clear restriction when addressing signalling events in mixed populations or subpopulations of cells. By combining flow cytometry with a panel of phosphospecific antibodies against several signal molecules simultaneously in a multi-parameter phospho flow cytometry analysis it is possible to obtain a higher level of understanding of the signal transduction dynamics at a single cell level. In addition, analysis of mixed cell populations makes it possible to study cells ex vivo in a state more closely resembling the in vivo situation. The multimeric analysis yields information on combinations of signals turned on and off in specific settings such as disease (signal nodes) that can be used for biomarker analysis and for development of drug screening strategies. Prostaglandin E2 (PGE2) is known to signal through four G-protein coupled transmembrane receptors, EP1–4, activating a multitude of potential signalling pathways. The analysis of the PGE2 signalling network elicited by activation of the four EP receptors in lymphoid cells revealing several signalling nodes is reviewed as an example.