(A) IgE traffics to lysosomes after entering DCs. DCs were incubated with 0.5 μg/ml hIgE-A647 for 4 hours before preparation for confocal microscopy. Data are representative of 17 images from two unique and representative donors. Original magnification, ×60; scale bars: 2.5 μm. (B) Effect of chloroquine on the intracellular IgE pool determined by confocal microscopy. DCs and basophils were incubated for 8 hours at 37°C with or without 0.5 μM chloroquine (Chlor) and stained with anti-IgE antibody before confocal microscopy. On each confocal micrograph, intracellular and cell membrane (extracellular) regions were identified manually (shown on the left; scale bar: 2.5 μm), fluorescence density was measured, and the signal ratio of intracellular to extracellular regions was determined. Shown are summarized data from 30 cells of each type in each condition for 2 donors. Data represent mean ± SEM and *P < 0.005. (C) Effect of chloroquine on intracellular IgE pool determined by flow cytometry. Basophils and DCs were incubated for 8 hours at 37°C with or without 2 mM chloroquine and washed with acid or PBS. The acid-resistant IgE fraction was determined by staining cells with anti-IgE after permeabilization and dividing the MFI of acid-washed cells by the MFI of unwashed cells as described for Figure 3B. Data are from 4 representative donors.