Activated MCs transfer internalized IgE–FcεRI to DCs through direct contact. (A) Maximum projection image from a confocal Z stack of a typical example of material transfer between an activated BMMC labeled with AF555-IgE (red) in contact with two immature BMDCs (seen in 36 of 45 cells counted across three independent experiments). Cells are fixed and labeled for actin (green) and nuclei (blue). Arrows indicate multiple occurrences of material transfer to each DC. The inset shows the actin labeling for the actMC, demonstrating actin clearance at the site of IgE–FcεRI accumulation. Image contrast in the red channel is enhanced to visualize the material transfer (puncta in the DCs) that is much lower in intensity than the MC-localized IgE. (B, top) The membrane marker GPI-GFP (green) transiently expressed in transfected BMMCs does not transfer to imDCs with the IgE-containing vesicles (red puncta). (bottom) When transiently expressed in transfected BMMCs, the endosomal marker, CD9 (green) does colocalize with the IgE-containing vesicles (red), both within the MCs and in those that have been transferred to the imDCs. White, boxed regions are enlarged and channel separated to the right of each merged image. Consistent results were acquired from two independent transfection experiments for each marker. (C) Representative images (from six independent experiments) of fixed labeling of actMCs co-incubated with imDCs. Cells were fixed after the indicated time and demonstrate colocalization of transferred material (IgE, red) with early endosomal compartments (EEA1, green), in the DCs (white boxes in merged image). The 10-min images (left) show an actMC in contact with a DC. The 30-min images (right) do not contain a MC, presumably because the interaction was lost by 30 min. All images were brightness and contrast enhanced. Bars, 10 µm.