Substrate-immobilized CCL21 increases the number and size of dynamic T-cell clusters. (A-D) Representative images of OT-II T cells (blue) and ovalbumin-loaded DCs (green) labeled before seeding with membrane-permeable dyes and cocultured on an uncoated substrate (A-B) or on a CCL21-coated substrate (C-D). After 72 hours of incubation, the majority of DCs were dead (red, propidium iodide in B,D), and large T-cell clusters were visible, especially in the CCL21-coated cultures (E-F; clusters marked with red arrows). Scale bars, 50 μm (A-D) and 200 μm (E-F). (G-H) Bar graphs illustrating the number (G) and total projected areas (H) of clusters formed on uncoated and CCL21-coated surfaces at different incubation times (data are representative of at least 3 independent experiments with 5 replicates each; error bars represent standard error of the mean; calculated P values [using t test] are as indicated in the figure). Note that substrate-bound CCL21 increased the number and projected areas of T-cell clusters. (I-J) Two examples of projected images of Z-stack series of single clusters formed on CCL21-coated surfaces (cluster borders are denoted by a yellow line). OT-II CD4⁺ cells, labeled before seeding with membrane-permeable dyes, green, blue, or red, were mixed in equal numbers and cocultured with antigen-loaded DCs on CCL21-coated surfaces for 72 hours. Note that the clusters are polyclonal, as evidenced by the fact that they contain cells tagged by all 3 colors. Scale bars, 20 μm. (K-P) Time-lapse frames depicting the dynamic properties of clusters (supplemental Video 1). As shown, clusters can merge; also, single cells can leave existing clusters and join new ones. Cluster merger events are color-coded as follows: red + pink (K, L) = dark green (M); dark green + light green (M) = light blue (N); light blue + dark blue (O) = purple (P). Scale bars, 50 μm.