The effects of CCL21 and ICAM1 on cell morphology are confined to functionalized areas along the substrate. (A) Experimental layout: silicon molds containing 4 separate, bottomless chambers were placed on a 35-mm glass-bottomed dish (A1). Different coating solutions were placed in each chamber to form separate strips containing no coating, ICAM1 coating, CCL21 coating, and CCL21+ICAM1 coating on the glass surface (A2). After overnight incubation, the coating solutions and silicon molds were removed (A3), and the surfaces were thoroughly rinsed. OT-II T cells were then cocultured with ovalbumin-loaded DCs (A4) and imaged by phase-contrast microscopy (supplemental Figure 2; supplemental Video 4). (B) A stitched image of the surface with cultured T cells, taken at 36 hours, in which each area is marked underneath the horizontal lines: no coating is marked in yellow, ICAM1 in blue, CCL21 in green, and CCL21+ICAM1 in purple. Magnified single fields are shown for each area (C-F). T cells are found mainly within the coated areas, which display a higher cell density (D-F) compared with that of the uncoated area (C). The induction of large clusters by CCL21 coating (B,E) and the cell spreading and culture flattening induced by ICAM1 (B,D,F) are confined to the coated area and do not affect the morphology of T cells in other areas that share the same original cell pool and culture medium. Scale bars, 500 μm.