pDCs activated in the presence of IL-3 with or without IL-10 express GrB mRNA and protein. PBMCs or purified pDC precursors (> 90%) were cultured for 16 hours in the presence of IL-3, either with or without IL-10. Freshly prepared unstimulated pDC precursors (0 hour) served as negative controls. (A) Zebra plots show GrB expression in pDCs from unfractionated PBMCs (top panel) or purified pDC preparations (bottom panel) as indicated by gating on the lin-1^...CD123^high populations. (B) Bar graphs indicate GrB expression as indicated by average median fluorescence intensities (MFI) from 6 independent experiments with purified pDCs. Error bars indicate SEM. (C) Fluorescence microscopy with ×60 magnification was performed with purified IL-3/IL-10–cultured pDCs stained with PE-labeled anti-GrB (orange) and FITC-labeled anti-BDCA-2 (green). (D) Spinning disk confocal microscopy was performed with purified pDCs cultured in the presence or absence of IL-3 and IL-10 and then stained with anti-GrB-biotin and streptavidin–Alexa Fluor 488 (green) as well as Cell Mask deep red membrane stain (red). The negative control shows staining with a biotinylated isotype control instead of anti-GrB-biotin. No green fluorescence was found when pDCs were cultured in the absence of IL-3 and IL-10 (not shown). (E) Line graphs and zebra plots show GrB expression by purified pDCs stimulated with IL-3 and IL-10 in the presence or absence of cycloheximide at 1 μg/mL (n = 3). (F) RT-PCR for GrB mRNA was performed using 6 × 10⁵ and 9 × 10⁵ purified pDCs isolated before (−) and after (+) incubation of pDCs for 16 hours with IL-3 and IL-10.