Figure 6 Multiparameter applications of phospho-specific flow cytometry. (A) U937 cells were treated with increasing amounts of IFN-g (from 4-1000 pg/ml) for 10 min at 37C. Cells were then split and lysed for Western analysis, or fixed and permeabilized for flow cytometry. The blot was probed with unlabeled pStat1, while pStat1 Ax488 or Ax647 were used for flow analysis. Note the large dynamic range (10,000 fold) covered by the FACS plots. Percent of maximum (using 1000 pg/ml as maximum induction) was calculated for all three analyses and plotted versus IFN-γ concentration. MFIs were used for flow cytometric data while integrated band intensities were used for the Western blot. (B) Multiparameter flow cytometric analysis of U937 cells. Cells were left unstimulated or treated with IFN-g (50 ng/ml) or IL-4 (10 ng/ml) and stained for pStat1 and pStat6. (C) Jurkat cells were left unstimulated or treated with PMA (50 nM) and ionomycin (1 mM) or anisomycin (2 mg/ml) and analyzed for pERK and pp38 levels. PMA/IO shows a strong pERK induction, as well as intermediate pp38 levels, while anisomycin leads to exclusive activation of pp38.