Figure 3. Diverse applications of phospho-specific flow cytometry. (A) Analysis of multiple stimuli. Jurkat T cells were treated with PMA or anisomycin and analyzed for phospho-p38 levels after formaldehyde fixation and methanol permeabilization (Ax = Alexa dye). The different levels of p-p38 produced by the two stimuli are indicative of their ability to induce p38 activity. Open histograms represent unstimulated cells, while filled plots indicate induced samples. (B) Inhibitor studies. Jurkat cells were treated with PMA and ionomycin (P/I), or with the MEK inhibitor U1026 prior to the addition of P/I. The inhibitor completely blocked phosphorylation of ERK, as one would expect from inhibition of the upstream kinase. (C) Dot plot layout. U937 cells were stimulated with IFN-γ and analyzed for phospho-Stat1 levels. This method of visualization allows two markers to be compared simultaneously and correlations to be drawn between the two. (D) Titration studies. U937 cells were treated with increasing amounts of IFN-γ, and measured for activation of Stat1. It is clear that titration data can be analyzed reliably by flow cytometry. Coupling titration experiments with inhibitor studies provides a novel platform for drug screening.