Figure 4. LZ B cells migrate within the... | Rockefeller University Press

Figure 4.

$Figure 4. LZ B cells migrate within the GC outskirts, whereas DZ B cells are highly confined to the GC. (A) LSFM images of popliteal LNs dissected from AID.Cre.tdTomato mice 12 d after immunization with NP-OVA. Different orientations of the same LN are shown in 3D volumetric and rainbow visualization mode. Representative images from five independent experiments with a total of 21 LNs from 12 mice. Scale bar, 200 μm. (B) LSFM 3D-rendered images of single GCs in different perspectives from AID.Cre.tdTomato (Cre) or AID.Cre.ERT2.tdTomato (Cre.ERT) mice that were immunized with NP-OVA. The graph indicates single-cell density across the GC. Magnification of top and bottom GC surfaces are shown in the right panels. Scale bar, 100 μm. Representative images of 70–212 GCs from 10 independent experiments with 4–12 mice per time point. Scale bar, 100 μm. (C) LSFM 3D-rendered images of a single GC from an AID.Cre.YFP mouse that was immunized with NP-OVA and 12 d after receiving NP-tdTomato 24 h before LN removal. Representative images of 96 GCs from three independent experiments with a total of four mice. Scale bar, 100 μm. (D) Time series showing position of B1-8hi B cells expressing active PA-GFP, at different time points and locations after LZ (top panel) or DZ (bottom panel) photoactivation. The LZ is labeled with NP-tdTomato (red) within GCs of popliteal LNs of immunized live mice that received PA-GFP+ B1-8hi B cells before immunization. Representative images of three mice from two independent experiments. Dotted lines depict the photoactivated area, and arrowheads indicate recently departed B cells from the photoactivated region. Scale bar, 100 μm. (E and F) Graphs showing numbers and percentages of active PA-GFP+ cells over time and position (quantification of cells in all the Z-stacks). Absolute cell number outside the GC over time (F) or the fraction of active PA-GFP+ cells out of the total photoactivated cells at time 0, found outside the GC (E). Analyzed data of three mice per group from two independent experiments. **, P < 0.01; ***, P < 0.0001; two-tailed Student's t test. a.u., arbitrary units.$

LZ B cells migrate within the GC outskirts, whereas DZ B cells are highly confined to the GC. (A) LSFM images of popliteal LNs dissected from AID.Cre.tdTomato mice 12 d after immunization with NP-OVA. Different orientations of the same LN are shown in 3D volumetric and rainbow visualization mode. Representative images from five independent experiments with a total of 21 LNs from 12 mice. Scale bar, 200 μm. (B) LSFM 3D-rendered images of single GCs in different perspectives from AID.Cre.tdTomato (Cre) or AID.Cre.ERT2.tdTomato (Cre.ERT) mice that were immunized with NP-OVA. The graph indicates single-cell density across the GC. Magnification of top and bottom GC surfaces are shown in the right panels. Scale bar, 100 μm. Representative images of 70–212 GCs from 10 independent experiments with 4–12 mice per time point. Scale bar, 100 μm. (C) LSFM 3D-rendered images of a single GC from an AID.Cre.YFP mouse that was immunized with NP-OVA and 12 d after receiving NP-tdTomato 24 h before LN removal. Representative images of 96 GCs from three independent experiments with a total of four mice. Scale bar, 100 μm. (D) Time series showing position of B1-8^hi B cells expressing active PA-GFP, at different time points and locations after LZ (top panel) or DZ (bottom panel) photoactivation. The LZ is labeled with NP-tdTomato (red) within GCs of popliteal LNs of immunized live mice that received PA-GFP⁺ B1-8^hi B cells before immunization. Representative images of three mice from two independent experiments. Dotted lines depict the photoactivated area, and arrowheads indicate recently departed B cells from the photoactivated region. Scale bar, 100 μm. (E and F) Graphs showing numbers and percentages of active PA-GFP⁺ cells over time and position (quantification of cells in all the Z-stacks). Absolute cell number outside the GC over time (F) or the fraction of active PA-GFP⁺ cells out of the total photoactivated cells at time 0, found outside the GC (E). Analyzed data of three mice per group from two independent experiments. **, P < 0.01; ***, P < 0.0001; two-tailed Student's t test. a.u., arbitrary units.