Figure 3.

BCR microclusters grow in FI with time when encountering antigen-containing lipid bilayers. (A–F) Shown are two-color time-lapse TIRF images of μ-High J558L cells labeled with Alexa Fluor 568–Fab anti-IgM placed on planar lipid bilayers containing antigen NIP1-His12 (A–C) or lacking antigen (D–F) over a time course of 120 s (Video 5). The BCR microclusters were examined by simultaneously imaging Igα-YFP (green) and Alexa Fluor 568–Fab anti-IgM (red), as described in Materials and methods. Bars, 1.5 µm. Typical microclusters in the images indicated by the white boxes are shown at 600% magnification for better resolution. The FIs of these microclusters were fitted by a 2D Gaussian function for precise 2D (x, y) coordinates and integral FI profiles, as detailed in Materials and methods. The normalized FI (B and E) and 2D trajectories by means of x versus y footprints (C and F) accumulated over the 120-s time course of these two typical microclusters are given. 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. The normalized FIs of all BCR microclusters analyzed by Igα-YFP (G) or Alexa Fluor 568–Fab anti-IgM (H) in μ-High J558L cells placed on lipid bilayers containing no antigen, NIP1-His12, or pNP1-His12 represent means ± SEM from 9–13 μ-High J558L cells in three independent experiments. For Igα-YFP clusters, data from six experiments were pooled.