Figure 4. Multi-dimensional analyses with phospho-specific flow cytometry. (A) Surface markers with phospho-epitope staining. Murine splenocytes were subjected to IFN-γ stimulation or left unstimulated, then stained with B220, TCR-b, and CD11b to distinguish B cells (blue), T cells (red), and monocytes/dendritic cells (green), respectively. The cell types were simultaneously analyzed for induction of Stat1 and Stat5 phosphorylation with phospho-specific antibodies. B cells and T cells showed clear Stat1 responses to IFN-γ, but the CD11b positive population was heterogeneous in its response. Only minor inductions of phospho-Stat5 are seen.  (B) Multiple kinases. U937 cells were treated with IFN-γ, IL-4, and IL-6 in the combinations shown. The cells were then analyzed for pStat1, pStat3, and pStat6 simultaneously after fixation and permeabilization. The top panel shows histograms of each channel individually and clearly shows the expected induction of Stat1 with IFN-γ, Stat3 with IL-6, and Stat6 with IL-4. When plotted in two dimensions (lower left panel), two samples appear coincidentally in the pStat1/pStat6 positive quadrant. However, when one analyzes these samples for pStat3, only the sample treated with IL-6 shows an induction. Therefore, samples that appear homogeneous within two dimensions can be separated clearly with simultaneous staining in three dimensions. Such correlations are not possible with Western blotting. The lower right panel is a representation of the data generated by a FACS analysis tool being developed in our laboratory. Each row represents a different stimulus, and each column represents a phospho-protein. The color of each block is indicative of the fold change in median fluorescence intensity in that channel. The data is easily visualized and compared without needing to plot all 15 samples. Larger screening experiments will require this form of analysis.