Lyn and Syk in the initiation of B cell spreading in response to membrane-bound antigen. (A–F) DT40 B cells were settled onto bilayers containing anti-IgM as antigen (Ag; red). (A) Lyn-KO and Syk-KO are shown. (top) DIC (left) and confocal microscopy (right) visualizing antigen. (middle) Confocal microscopy visualizing B cell membrane (PKH26). (bottom) IRM. Bars, 5 μm. (B) Quantification of (left) contact area by IRM and (right) antigen accumulation. (C) Two-dimensional tracking of individual WT, Lyn-KO, and Syk-KO cells. (D) SEM images. Bars, 2 μm. (E) TIRFM images of GFP-Syk (green) expressed in reconstituted Syk-KO and Lyn-KO. Relative fluorescence intensity plots to indicate the distribution of antigen and GFP-Syk are depicted by the diagonal dashed lines. Bars, 5 μm. (F) Quantification of GFP-Syk–containing antigen microclusters present after 3 min of interaction in reconstituted Syk-KO and Lyn-KO DT40 B cells. Data are from two experiments, and the number of antigen microclusters containing GFP-Syk was counted in 24–37 cells, representing a total of 698 microclusters. The mean numbers for reconstituted Syk-KO and Lyn-KO are 29 ± 2 and 0, respectively. ***, P < 0.0001. Video 3 is available.