Figure 2.

Substrate-immobilized CCL21 + intercellular adhesion molecule 1 (ICAM1) increase cytotoxic T-cell number and proliferation. (A–D) Representative fluorescence images of T-cells grown on different coated substrates for 72 h (A,B) or 7 days (C,D), following the breakdown of cell clusters, and their spin-down. Cell nuclei are stained blue in all cells, and red only in dead cells. Scale bar: 50 µm. (E–H) Viable cell numbers and percentage of dead cells at 72 h (E,G, respectively) and 7 days (F,H, respectively), quantified using automated image analysis. Data are from one experiment representative of at least three independent experiments with 20 replicates each (see Figure S1A in Supplementary Material). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure. The number of T-cells seeded per well was 3 × 10³. CCL21 and ICAM1 coatings collectively increase viable cell numbers by up to ninefold, without significantly affecting cell death. (I,J) Histogram and bar graph illustrating the increase in cell proliferation induced by CCL21 + ICAM1, indicated by a decrease in the mean fluorescent intensity of CFSE, compared to cells grown on the uncoated culture (data are representative of three independent experiments with four replicates each). Error bars represent SEM. Calculated p-values (using standard t-test) are as indicated in the Figure]. Histograms in (I) show CFSE levels: green—non-activated T-cells; black —T-cells activated on uncoated substrates; blue—T-cells activated on CCL21 + ICAM1-coated substrates.