Multiplex detection of secreted cytokines. Secreted cytokines and chemokines following co-culture of CD4+ T cells with peptide or mock pulsed B cells were detected by multiplex assay. (A) and (B) Cytokine levels were tested in CD4+ T cell supernatants at serial time points over 18 hours with data for IFN-γ and TNF-α shown for two experiments (Ant treatment was run once). (C) Total area under the curve for secretion of each cytokine over 4 hours post-stimulation was calculated as described in Materials and Methods. Mean values from two replicate experiments are shown; error bars show SEM. (D) Percent suppression of agonist-induced cytokine suppression by addition of antagonist peptide. Suppression was calculated based on the AUC of cytokines four hours after stimulation. Dashed lines indicated the 95% confidence interval of the mean of 2 experiments, error bars show SEM.