Phosflow protocol for human pbmcs

The phosphorylation of ERK1/2 was analyzed using the BD PhosFlow Protocol for Human PBMCs. After isolation, PBMCs were incubated for 30 min in complete medium (Table S5) without or with DARA at 1 µg/ml. Afterwards, the samples were washed and resuspended in complete medium and left to equilibrate at 37°C for 20 min. An equal volume of prewarmed complete medium containing either α-Ig, CpG-B, or IL-2 (as described in Materials and methods) were added and the samples were incubated for 10, 30, or 60 min at 37°C, or left unstimulated (control). The samples were then fixed with BD Cytofix Fixation Buffer (BD Biosciences) for 10 min at 37°C, washed twice in Phosflow Perm/Wash Buffer I (BD Biosciences), and stained for pERK1/2 (Table S4) for 60 min at room temperature. Surface and intracellular stainings with other mAbs were performed as required by the experimental design. Samples were washed and resuspended in Phosflow Perm/Wash Buffer I for flow cytometric analysis.