Fig. 1

Phosphoflow cytometry method. A heterogeneous population of cells is briefly stimulated with cytokines, which bind to receptors and phosphorylate intracellular signaling proteins A. Cells are then fixed to preserve their phosphorylation status and stained with fluorescently labeled extracellular antibodies to define cell populations. Next, cells are permeabilized to allow access to intracellular proteins and stained with fluorescently labeled intracellular antibodies that specifically recognize phosphorylated forms of proteins of interest B. Labeled cells are then detected by flow cytometric analysis to determine phosphorylation levels in specific immune cell subsets of cytokine-stimulated and unstimulated groups C. MFI, mean fluorescent intensity. P-STAT, phosphorylated signal transducer and activator of transcription