Intracellular cytokine optimization and standard operating procedure.

We describe here a method for optimizing the use of polychromatic flow cytometry (with up to 17 fluorochromes simultaneously) in surface and intracellular staining of human T lymphocytes. We will highlight and discuss how to procedurally optimize key steps in the experimental process before an intracellular cytokine staining assay protocol is finalized. These include but are not limited to the titration of monoclonal antibodies, use of a dead-cell discriminator and 'dump' channel, selection of a cytokine secretion inhibitor, selection of fixation and permeabilization reagents, and inclusion of compensation controls. Building on this basic protocol, we then establish a polychromatic assay designed to detect five separate functions of T lymphocytes (production of three cytokines and one chemokine, and degranulation) while simultaneously identifying multiple surface markers on the responding cells.