Flow Cytometric Detection of p38 MAPK Phosphorylation and Intracellular Cytokine Expression in Peripheral Blood Subpopulations from Patients with Autoimmune Rheumatic Diseases

Figure 5

Optimal cytokine expression and p-p38 detection within PBMCs subsets permeabilized with methanol. PBMCs were stimulated with PMA plus Ionomyci at various time points, permeabilized in methanol, and stained with MoAbs against CD3, CD56, CD11c, p-p38, and IFN-γ. CD56+ cells were subgated and the percentage of CD11c+ within CD56+ cells is indicated. Gated CD56+ cells were plotted for CD3 versus IFN-γ and CD3 versus p-p38. As it can be seen, IFN-γ expression is detectable between 60′ and 240′, reaching its peak at 120′, while maximal p-p38 activity is better visualized at 30′. NKT cells express more IFN-γ than NK as indicated by the measured percentages and mean fluorescence intensity of IFN-γ+ NKT cells (see text for more details).