Immobilized CCL21 and ICAM1 increase viable cell numbers of antigen-specific T cells. (A-H) Representative fluorescent images of T cells grown on different coated surfaces after the breaking down of clusters and their spin-down. Images were taken after 72 hours (A-D) and 7 days (E-H) of culture. Cell nuclei are stained blue in all cells, and red only in dead cells. Scale bars, 50 μm. (I) Bar graph illustrating viable cell numbers, as quantified using automated image analysis after cluster breakdown, and their spin-down (data are representative of at least 3 independent experiments with 3 to 5 replicates [wells] and 20 fields of view in each well; see supplemental Figure 3 for summary of all independent tests). Error bars represent standard error of the mean. Immobilized CCL21 or ICAM1 alone doubled live cell numbers at all time points. Combining the 2 further increased the yield of live cells from 48 hours onward, with a more than fourfold increase at 7 days. (J-K) Representative fluorescent images of OT-II T cells (ova specific) expressing ubiquitin-green fluorescent protein (green), and non–ova-specific T cells, mixed at a 1:99 ratio. Cells were cultured with ova-loaded DCs for 7 days on an uncoated surface (J) or on a surface coated with CCL21+ICAM1 (K). Images were taken after the breaking down of clusters and their spin-down, facilitating automated cell counting. Cell nuclei are stained blue, dead cell nuclei are in red (propidium iodide). Scale bars, 50 μm. (L) Bar graph illustrating the ratio of viable OT-II T cells to the originally seeded cell number, as quantified by automated image analysis (Data are representative of at least 3 independent experiments with 6 replicates (wells) and 20 fields of view in each well. Error bars represent standard error of the mean). Coating with CCL21+ICAM1 enriches specifically activated T cells (see supplemental Figure 4 for data of non-ova T cells). Calculated P values in panels I and L (using t test) are as indicated in the figure.