Comparison of CD4⁺ T-cell chemotaxis in response to media from influenza vaccine-stimulated LN lymphocytes and FVIII-stimulated splenocytes. Chemokine gradients were generated by stimulating splenocytes and LN-lymphocytes with FVIII (1 μg/mL) or influenza with and without MF59 (1 μg/mL) for 18 hours. CD4⁺ T-cell chemotaxis competition of between converging gradients were imaged overnight between FVIII and influenza (A) and FVIII and influenza with MF59 (B). (C) Media from unstimulated splenocytes and LN-lymphocytes were assessed as a negative control. (D) A CCL5 (20 μg/mL) gradient was assessed as a positive control. The vertical components traveled by T cells were analyzed for FVIII and influenza competition (E) and FVIII and influenza with MF59 (F). Statistical analysis was conducted using Rayleigh’s test and Mann-Whitney U test for trajectory plots and distance traveled, respectively. Data representative of at least 3 independent experiments. *P < .05; **P < .01; ***P < .001.