The duration of APC-T contact does not correlate with APC effectiveness. Naive DO11.10 T cells were embedded in 3-D collagen matrices together with different APCs, labeled or not with OVA peptide. (A) Cell movements were recorded by time-lapse video microscopy, and the duration of individual T cell-APC contacts was analyzed. Each dot represents one observed contact. Bars represent the median. Dots at more than 60 minutes are cell pairs that have been observed for more than 60 minutes without releasing the contact. Data are pooled from 4 independent experiments (or 2 experiments, for activated B cells). (B) T-cell proliferation within such gels was analyzed by CFSE down-regulation after 72 hours of coincubation in the presence of antigen followed by collagenase digestion of the gels. The numbers indicate the percentage of cells that have undergone one or more divisions. (C) The kinetics of T-cell proliferation was studied by CFSE-dilution analysis. To this end, CFSE-labeled DO11.10 T cells were embedded together with APCs in 3 individual gels at time point 0. After 24, 48, and 72 hours gels were digested and analyzed for dilution of CFSE. All data were gated on CD4⁺ cells, which were identified by simultaneous staining with an anti-CD4 antibody. Open symbols indicate APCs without OVA; closed symbols, APCs with OVA. Data are representative of 5 experiments performed (activated B cells 2).