Cytometry of intracellular signaling: from laboratory bench to clinical application

The analysis of signaling pathways based on combinations of phospho-specific antibodies is now a well-recognized flow cytometry technique. Despite its wide-ranging potential in the fields of biology, industry, and medicine, it has been relatively slow to gain widespread use, and is often considered to be technically challenging. In this chapter, we detail protocols developed in our laboratory for monitoring signaling pathways in blood samples based on combinations of phospho-specific antibodies. Emphasis is placed on clinical application. The assays have a modular design, with a core protocol for whole blood fixation and lysis, a suite of agents that can acutely activate or inhibit the different signaling pathways, and a wide range of phospho-specific antibodies as the readout.