TLR4 ligand–stimulated B cells that have entered the dark zone can compete efficiently with antigen-specific B cells for dark zone entrance and can divide and persist in the spleen. (A) Competition. Fluorescently stained HEL-transgenic B cells (blue) and LPS-pulsed B cells (green) were adoptively transferred into a recipient mouse immunized 10 d previously with HEL. FDC-M2 antibody was injected 1 d later, and the next day intravital TP-LSM intravital imaging was performed. An MIP image from TP-LSM intravital imaging of the iLN is shown on the left. The tracking of LPS-stimulated and transgenic B cells is shown on the right. The location of the B cells at the beginning of the tracking is shown with a green (LPS stimulated) or blue (not stimulated) ball. Bar, 100 µm. (B) B cells dividing in the dark zone. MIP from intravital TP-LSM imaging of the dark zone of a germinal center showing three cells that divided during 20 min of imaging. White squares show three cells in the dark zone that divided during a 45-min imaging. Zoomed images from the TP-LSM imaging, which are focused on the cell outlined in yellow in the left, are shown on the right. The arrows indicate cell dividing. Bar: (left) 40 µm; (right) 50 µm. (C) Persistence in the spleen. TP-LSM images of cryostat sections of spleen. Fluorescently stained B cells (blue) and LPS-stimulated GFP-B cells (green) were adoptively transferred into the recipient mouse immunized 7 d previously with HEL. 4 d later, 10 µg of labeled anti–FDC-M2 antibody (red) was injected subcutaneously. The following day, spleen was collected, fixed, and cryostat sections prepared. The image on the left was acquired with a 10× lens and the image on the right was acquired with a 40× lens. GC, germinal center. Bar: (left) 100 µm; (right) 50 µm. The experiment was performed twice with similar results.