Vav is required for B cell spreading. (A–F) B cells were settled onto bilayers containing anti-IgM and HEL as antigen (Ag) for DT40 and primary B cells, respectively. (A) Vav3-KO DT40 B cells. (top) DIC (left) and confocal microscopy (right) visualizing antigen. (middle) Confocal microscopy visualizing B cell membrane (PKH26). (bottom) IRM. Bar, 5 μm. (B) WT, Vav3-KO, Vav3-KO expressing Vav-GFP, and Vav3-KO expressing Vav with mutated GEF activity DT40 B cells. Quantification of (left) contact area by IRM and (right) antigen accumulation. (C) SEM images. Bars, 2 μm. (D and E) Primary MD4-HEL-Tg (WT) and Vav1/2-KO HEL-Tg (Vav1/2-KO) splenic B cells. (D, top) Confocal microscopy visualizing antigen. (bottom) IRM. Bars, 2.5 μm. (E) Quantification of (left) contact area by IRM and (right) antigen accumulation. (F) TIRFM images of DT40 B cells expressing Vav-GFP (green) after 3 min. Bar, 5 μm. A relative fluorescence intensity plot to indicate the distribution of Vav-GFP (green) and antigen (red) is depicted by the diagonal dashed line. Video 9 is available.