Substrate-immobilized ICAM1, with or without CCL21, reduces T-cell clustering and induces their spreading on surfaces. (A1-C2) Representative scanning electron micrographs, showing T-cell clusters formed in cultures without surface coating (A1, A2) and in cultures with CCL21 coating (B1, B2) or CCL21+ICAM1 coating (C1, C2). Note that ICAM1 coating (C1, C2) induces cell spreading, resulting in significantly smaller and flatter clusters. Scale bars in A1-C1, 100 μm; in A2-C2, 10 μm. (D-F) Transmitted light microscopy images, depicting cell spreading and a decrease in cluster size after their culturing for 72 hours on a surface coated with ICAM1+CCL21 (E), compared with CCL21 alone (D). Addition of anti-LFA1 blocking antibodies to the culture medium of cells on CCL21-coated surfaces inhibits cluster formation (F). Scale bars, 200 μm; white squares show enlarged area in which the scale bar is 100 μm. (G-J) Representative phase contrast images, showing cells 83 hours after their seeding on coated substrates. Note that the ICAM1 surface coating modifies single-cell morphology. Nonclustered cells on an uncoated surface are mostly round, with hardly any cell–cell contacts (G), whereas on CCL21-coated surfaces, cells commonly display an elongated morphology (H). On ICAM1-coated surfaces, the majority of the cells are elongated, with multiple cell–cell contacts (I, J). Scale bars, 50 μm (supplemental Video 2).