Human peripheral blood mononuclear cell culture for flow cytometric analysis of phosphorylated mitogen-activated...

Lymphocyte activation and fine tuning of downstream signaling circuits for the regulation of cytokine expression are critical for a successful immune response. Hence, technical protocols permitting simultaneous testing of these attributes in peripheral blood lymphocytes are of paramount importance. Phospho-specific flow cytometry is a novel methodology that detects phosphorylation of signaling effectors in multiple, rare cellular populations within peripheral blood. In addition, it allows the quantification of phosphorylation levels for signaling proteins within each single cell, and therefore is superior compared to traditional biochemical approaches, such as Western blotting. One such important signaling pathway within immune cells is the p38 MAPK pathway involved in the regulation of cytokine expression, cell proliferation and apoptosis. In this chapter, we provide technical instructions for culturing human peripheral blood lymphocytes for simultaneous monitoring of p38 MAPK phosphorylation and associated cytokine expression, especially in rare cell populations, such as NK and NKT.