The growth of BCR microclusters is selective and antigen concentration dependent. (A–F) Two-color time-lapse TIRF images of Igα-YFP (green) and NIP1-His12-Hylight647 (red) are given at the indicated time points (Video 6) for μ-High J558L cells placed on planar lipid bilayers containing NIP1-His12-Hylight647 (A). Similarly, two-color time-lapse TIRF images of Igα-YFP (green) and Cy3–Fab anti–MHC I (red) are given at the indicated time points (Video 7) for μ-High J558L cells placed on planar lipid bilayers containing NIP1-His12 (D). Bars, 1.5 µm. (A and D, bottom) Typical microclusters magnified 600% and analyzed by 2D Gaussian fitting. Shown are the normalized FI for Igα-YFP and NIP1-His12-Hylight647 (B) and Igα-YFP and Cy3–Fab anti–MHC I (E). The gray horizontal lines in B and E show the background FI values for these two typical microclusters over time. The background FI value is the Z₀ value acquired in the 2D Gaussian function upon mathematical fitting, as shown in Fig. S4 A. Normalized FI of all Igα-YFP and NIP1-His12-Hylight647 clusters (C) or Igα-YFP and Cy3–Fab anti–MHC I clusters (F) are given with time. (G–N) The normalized FI (G–J) and size (K–N) of microclusters examined by Igα-YFP (G and K), Alexa Fluor 568–Fab anti-IgM (H and L), NIP1-His12-Hylight647 (I and M), or Cy3–Fab anti–MHC I (J and N) are given for μ-High J558L cells placed on lipid bilayers lacking antigen or containing NIP1-His12 antigen at a concentration of 10 nM (25 molecules/µm²) or 50 nM (100 molecules/µm²). The data represent means ± SEM of the indicated numbers of clusters in three independent experiments.