Absence of a long- or short-range CXCL13-mediated directional B-cell migration into B-cell follicles. (A) Schematic outline of the normalization protocol to pool tracks from various 2-P microscopy image sequences. (B) Normalized tracks of WT (left panel) and CXCR5^−/− (right panel) B cells migrating in the T-cell area. A total of 162 WT and 106 CXCR5^−/− B-cell tracks are pooled and presented such that the closest intersection point with the nearest B-cell follicle at the onset of cell tracking is at the 12 o’clock position. The percentage of tracks falling into a 120° angle toward B-cell follicles (dashed lines) is indicated. (C) Example of 2-P microscopy image at indicated times post B-cell transfer. Lines define B-cell follicles. The dotted squares are shown enlarged in D and E. Scale bar, 50 µm. (D) A 2-P microscopy image of B cells migrating parallel to and away from an adjacent B-cell follicle taken from the dotted outline from the left panel in C. The top panel shows the starting point of the B cells at the onset of the recording, adjacent to a B-cell follicle. Time in minutes and seconds. (E) A 2-P microscopy image of a B-cell track migrating into an adjacent B-cell follicle taken from the dotted outline from the middle panel in C. (F) Frequency of WT B-cell tracks either entering or moving parallel to/away from adjacent B-cell follicles. All tracks (n = 23) were recorded within the first 150 minutes after B-cell transfer between 20-µm and 130-µm depth. Fo, B-cell follicle.