DT40 B cells as a model system to examine cellular responses to membrane-bound antigen. (A–C) DT40 B cells were settled onto bilayers containing anti-IgM as antigen (Ag). (A, top) DIC (left) and confocal microscopy (right) visualizing antigen. (A, middle) Confocal microscopy visualizing B cell membrane (PKH26). (A, bottom) IRM visualizing contact area. Bar, 5 μm. (B) Bilayer antigen density was varied, as depicted, and (left) contact area by IRM and (right) antigen accumulation were quantified. (C) SEM images. Bar, 2 μm. (D and E) DT40 B cells expressing GFP-Syk (green) were settled on bilayers containing anti-IgM (red) as antigen (Ag). (D) TIRFM images. Relative fluorescence intensity plots of GFP-Syk and antigen are depicted by the diagonal dashed lines. Bar, 5 μm. (E) Quantification of GFP-Syk–containing antigen microclusters after 3 min at the indicated antigen densities. Data represent two independent experiments. The number of antigen microclusters containing GFP-Syk was counted in at least 15 cells of each cell type, representing a total of 779 microclusters. Mean numbers for high and low antigen density are 25 ± 2 and 17.4 ± 1, respectively. **, P < 0.005. Videos 1 and 2 are available.