Flow cytometric analysis of cell signaling proteins

In recent years, techniques that combine the use of phospho-specific antibodies and multiparameter flow cytometry have been developed for the detection of protein phosphorylation at the single cell level. Flow cytometry is uniquely suited for this type of analysis, as it can measure functional and phenotypic markers in the context of complex cell populations. Phosphorylation can be assessed simultaneously in multiple cell subsets, and due to the small sample sizes required, and the rapid analyses of large numbers of cells in this approach, rare cell analysis is possible without the ex vivo expansion of cells.In this chapter, we detail flow cytometric protocols for the detection of intracellular phospho-proteins in samples derived from whole blood and peripheral blood mononuclear cell preparations. These protocols define steps for cell activation, fixation, permeabilization, and staining by phospho-specific and phenotyping antibodies. We discuss technical difficulties inherent to this technique and suggest solutions to commonly encountered problems. Additionally, we show examples of phospho-protein detection in lymphocyte subsets, dendritic cells, and monocytes activated with various stimuli, including mitogens, cytokines, and superantigens. Finally, we highlight a potential clinical trial application for this flow cytometric assay as a platform for pharmacodynamic monitoring of kinase inhibitors.