Figure 3. Phospho-staining in 2 and 3 dimensions.

A) 1X106 CD4+ na´ve T cells (purified by negative isolation) were treated with IL-4 or IL-12 (200 ng/ml) for 12 hours. Cells were fixed, permeabilized, and stained (as per protocol 2) and stained with phospho-STAT6(Y641)-AX488 (clone 18) and phospho-STAT1(Y701)-AX647 (clone 4a). Antibodies were used at 0.25 mg. B) Three MAPK kinase signaling responses simulataneously. Human PBMC depleted for adherent cells were cultured in either 10% autologous human sera or 10% FCS (Hyclone). Cells were stained for phospho-p44/42(T202/Y204)-AX488 (clone 20a), phospho-p38(T180/Y182)-PE (clone 36), and phospho-JNK(T183/Y185)-AX647 (clone 41) as per protocol 1. Antibodies were used at 0.125 mg (p-p44/42) and 0.25 mg (p-p38 and p-JNK).