Cognate interactions of DO11.10 T cells with B cells within a 3-D collagen gel and in a lymph node in situ are long-lasting. Naive B cells or mature DCs were loaded with 100 μg/mL OVA peptide and embedded within a 3-D collagen matrix together with freshly prepared DO11.10 T cells. B cells had been labeled with CFSE or SNARF before embedding. Cell movements were recorded by time-lapse video-microscopy. (A) Formation and migration of a T cell-B cell pair (Supplemental Movie 1). The white line indicates the migration path taken over time. The B-T pair is stable over the entire observation period. For better visibility the relevant B-T pair has been enlarged by 50%. Five hours after culture set-up. (B) Interaction of a T cell with a DC (Supplemental Movies 3 and 4). Note that the interaction lasts only for 13 minutes. Twenty-five hours after culture set-up. (C) T cells receive a calcium signal on cognate interaction with naive B cells. A T cell is shown, which fluxes intracellular calcium while moving around a spot of contact with a naive B cell (Supplemental Movie 2). Thirty minutes after culture set-up. (D) SEM analysis of a cognate B-T pair fixed in the process of migration within 3-D collagen. (E) As in panel D, for a cluster of 3 T cells and 1 DC. B cells were stained with CFSE and injected into a Balb/c mouse together with cell tracker orange-stained DO11.10 T cells. Intravital imaging was performed in the inguinal lymph node. (F) A T-B pair migrating within the B cell follicle. Note the similarity of morphology as compared with the appearance in 3-D collagen. The path of the cell pair during migration is indicated (Supplemental Movie 11). Four hours after injection of T cells. (G) Multiple T-B pairs form at the T zone border and within a B-cell follicle (arrowheads, Supplemental Movie 12). Seven hours after injection of T cells. Scalebar: 100 μm (G), 50 μm (A,B,F), 10 μm (C-E). Numbers in panels A-C and F indicate minutes of real time.