BafA prevents the localization of small molecule TLR7 agonists to the MIIC. pDCs, precultured overnight with IL-3, were treated with BafA (100nM) for 2 hours before stimulation with SM-AF488 at 3 μm (A), R837-TAMRA at 50 μm (B), or CpG-B–TAMRA at 10 μm (C). After 90 minutes of stimulation, cells were fixed, permeabilized, and stained with anti–HLA-DR-APC and purified rabbit polyclonal anti–LAMP-1 Ab followed by anti–rabbit Ab labeled with AF568 (A) or with AF488 (B-C). Images were acquired with a ZEISS LSM 710 confocal microscope and an oil-immersion objective (63×/1.4 NA), with the pinhole set for a section thickness of 0.8 μm (pinhole set to 1 airy unit in each channel). Images were acquired sequentially with separate laser excitations to avoid cross talks between different fluorophores and processed with Zen2008 software. Images show several cells from 1 representative donor of 3. Cells in squares are shown in higher magnification on the right. (D) After 18 hours of stimulation with R837-TAMRA (50 μm) or CpG-B–TAMRA (10 μm), compound uptake by pDCs was evaluated by flow cytometry.