Fig 1: Effect of mechanical confinement on T cell activation. Protein expression of activation markers on T cells of (a) CD69 (24 hours after activation), (b) IL-2 (24 hours after activation) and (c) CD25 (4 days after activation). The highest upregulation of proteins was observed for cells confined in 10 μm high channels, whereas confinement in 5 μm high channels results in downregulation of the corresponding proteins. No significant change was observed for cells passing through 15 μm high channels relative to the control (inlet). Importantly, only live cells were analyzed for activation markers, indicating that the decreased activation seen in the 5 μm condition reflects an active phenotype of surviving cells, rather than an artifact of dead cells. All box diagrams present data pooled from six independent experiments performed in replicates with the horizontal line representing the mean value. Statistical comparisons between these samples were performed using a two-sided Wilcoxon signed-rank test. P-values of ns, *, and ** correspond to p > 0.05, p < 0.05, and p < 0.01, respectively.