The number of single B cells located next to individual GC structures is highly variable. (A) LSFM 3D-rendered images of an LN removed from an immunized AID.Cre.tdTomato mouse (top left), and analysis for separate GCs (each GC is depicted in a different color) and individual B cells within 50 μm from each GC (top right). Higher magnification images of two GCs from the same LN, indicated by I and II, demonstrating volumetrics, proximal cells, and surface roughness analysis. Representative images from 5 independent experiments with a total of 21 LNs from 12 mice. Scale bar, 50 μm. (B) Quantification of the number of cells detected at a 50-μm distance from the GC, normalized to the GC surface area (proximity index) over time in AID.Cre.tdTomato mice. Data pooled from 3–5 independent experiments with a total of 4–21 LNs from 3–12 mice per group. (C) Correlation plots of roughness index (Materials and methods) and proximal index as measured for two representative single LNs. The R2 values represent coefficients of determination. (D) LSFM 3D-rendered images of an LN removed from an immunized AID.Cre.ERT2.tdTomato mouse as in A that was treated with tamoxifen. Representative images from two independent experiments with eight LNs from four mice. Scale bar, 50 μm. (E) Quantification of proximity index as in B for GCs in AID.Cre.tdTomato (Cre) and AID.Cre.ERT2.tdTomato (Cre.ERT) mice 14 d after immunization with NP-OVA. Data pooled from two independent experiments with eight LNs from four mice per group. ***, P < 0.0001; two-tailed Student's t test.
