ICAM1-coated surfaces, with or without CCL21, decrease culture height. (A-C) Transmission electron microscopy images of T-cell clusters formed on surfaces coated with CCL21. The experiment demonstrates that clusters are loosely packed, despite multiple membrane contacts between cells within the cluster. Scale bars, 5 μm (A), 1 μm (B), and 0.5 μm (C). (D-H) Transmission electron microscopy images of transverse sections of T cells, cultured on a CCL21+ICAM1-coated surface (surface marked with red arrows in panel D). Note that the T cells formed multiple, noncontinuous membrane contacts, both with the coated surface (D-E,H) and with neighboring cells (D,F-G). Scale bars, 5 μm (D), 200 nm (E), 500 nm (F), 200 nm (G), and 500 nm (H). (I1-L2) Representative fluorescent Z-stack projected images of single T-cell clusters, formed on different coatings, are shown in top views (I1,J1,K1,L1) and in matching side views (I2,J2,K2,L2). T-cell membranes are live-stained green, and cell nuclei are live-stained red (supplemental Video 3). (M) Culture heights were calculated on the basis of Z-stack deconvolution imaging (data are representative of at least 2 independent experiments with 10 replicates each; error bars indicate standard error of the mean; calculated P values [using t test] are as indicated in the figure). Note that clusters formed on surfaces coated with ICAM1, either with (L1,L2,M) or without (K1,K2,M) CCL21, are thinner compared with those formed on uncoated surfaces (I1,I2,M), whereas very large and thick clusters are induced by CCL21 coating (J1,J2,M). Scale bars, 20 μm.