ECM is essential for Th1 migration. (A) The percentage of mobile versus immobile Th1 cells in confinement on ICAM-1, FN, PLL, and passivated surface in serum-free media. Mobile cells are defined by a minimum displacement of 10 µm. (B and C) Analysis of the (B) effective velocities and the (C) actual displacement of mobile cells in A. (D) Cell tracks on passivated substrates versus on FN in either serum-free media (−FBS) or with serum (+FBS). The colors represent the cell’s average velocity. (E and F) Effective velocities (E) and actual displacement (F) of cells pretreated for 3 h with a mixture of integrin-blocking antibodies compared to control (PBS). (G and H) Tracking of cells and representative trajectories of cells on (G) passivated versus (H) FN-coated substrates in serum-free media. The cell outlines are color-coded for time. Number of biological replicates (colored dots): (A–C) PLL n = 3; Passivated n = 4; 0.1 µg/ml FN n = 3; 1 µg/ml FN n = 3; 10 µg/ml FN n = 4. (E–F) PBS n = 4; Ab-integrin n = 4. Each replicates represent the average of 5–10 fields of view (gray dots = average of each fields of view), which each contain ∼250 cells. Statistical tests: (A–C) Two-tailed unpaired parametric t test; (E and F) One-tail unpaired parametric t test.