The affinity-dependent growth of BCR microclusters correlates with the strength of signaling. (A and B) μ-High and μ-Low J558L cells were fixed 10 min after placing them on lipid bilayers containing NIP1-His12 antigen. (C and D) B1-8 primary B cells were fixed 10 min after placing on lipid bilayers containing high affinity antigen NIP1-His12 or low affinity antigen pNP1-His12. The fixed B cells were probed for the recruitment of pSyk into the contact area of the B cells with the lipid bilayer by intracellular staining for pSyk. Shown are two-color TIRF images for BCR and pSyk (A and C). Bars, 1.5 µm. (B and D) The number of pSyk clusters accumulated in the contact area (top), the size of the contact area (middle), and mean BCR FI of the contact area (bottom). Each dot represents one cell analyzed in three independent experiments, and bars represent means ± SD. Two-tailed t tests were performed for the statistical comparisons in B and D. DIC, differential interference contrast.