BCR microclusters grow in both size and FI with time when encountering antigen-containing lipid bilayers. (A and B) Pseudocolor 2.5D Gaussian images of one typical Igα-YFP or Alexa Fluor 568–Fab anti-IgM microcluster are shown at the indicated times (Video 5) for μ-High J558L cells placed on planar lipid bilayers containing NIP1-His12 (A) or lacking antigen (B). The FWHM of each BCR microcluster upon 2D Gaussian fitting was used as a measure of the size of the microclusters, as detailed in Materials and methods and Fig. S4 A. Bars, 1.5 µm. (C and D) Means ± SEM of the normalized size of BCR microclusters analyzed by Igα-YFP (C) or Alexa Fluor 568–Fab anti-IgM (D) are given at the indicated time points for μ-High J558L cells placed on planar lipid bilayers containing NIP1-His12 or lacking antigen. Data represent the indicated numbers of BCR microclusters examined in three independent experiments. (E and F) Linear regression analyses of the FI and the size of BCRs. The slopes of the linear fitting for μ-High J558L cells placed on planar lipid bilayers containing NIP1-His12 antigen were 85 (Igα-YFP; E) and 106 (Alexa Fluor 568–Fab anti-IgM; F). The slopes for μ-High J558L cells placed on planar lipid bilayers containing no antigen were each 27 (E and F).