Th1 cell activation protocol and serum-free migration dynamics. (A) Graphical representation of Th1 activation. (B) Flow cytometer analysis of a representative single-cell suspension on the day of extraction from OTII mice using CD4 and Vα2 antibodies as markers. (C) A representative flow cytometer analysis of T cells three days post activation with anti-IL-4 (11B11; 40 µg/ml), IL-2 (20 U/ml), IL-12 (40 ng/ml), OVA peptide (4 µg/ml). T cells were isolated using lymphocyte separation media. (D–F) Representative flow cytometer analyses of Th1 T cells after 5 (D), 7 (E), and 9 (F) days post activation, with continuous treatment of 10 units/ml IL-2 and lymphocyte separation media before each use. (G and H) The actual displacement (G) and effective velocities (H) of mobile cells on fibronectin and ICAM-1 substrate with and without FBS in cell media. (I) Th1 cell tracking in serum-free media on Fibronectin in unconfined versus confined (5 µm) environments. The colormap represents the cell average velocity in µm/sec. Number of replicates for G and H: All conditions n = 3. Samples with and without FBS were performed with the same preparation of cells, on the same day, from the same mice. Each replicate represents the average of 5–10 fields of view (gray dots = average of each field of view), which contain ∼250 cells. Statistical test: one-tail paired parametric t test.