Figure 2. General phospho-protein staining technique for flow cytometry. Cells containing three proteins, A, B, and C, are treated with a stimulus that leads to the phosphorylation of protein A (Stimulus A), protein B (Stimulus B), or both simultaneously. The cells are then fixed, permeabilized, and stained with fluorophore-conjugated phospho-specific antibodies to the active forms of proteins A and B (a-pA and a-pB). When cells are analyzed by flow cytometry the individual inductions cause increases in fluorescence on one fluorescent channel, i.e. in the green or red channel, while the dual stimulation causes in shift in both channels. This technique can also be applied to patient samples to help characterize aberrant signaling events that occur during disease progression, or determine the efficacy of signaling pathway-specific drugs in vivo. In this case, samples must be isolated from patients and immediately subjected to fixation and permeabilization conditions that will maintain phospho-epitope integrity.