Analysis of mobility and directionality of neutrophils migrating on substrates of different stiffness. (A) The MSD is plotted as a function of time between steps. Neutrophils on the softer gels (5 and 10 kPa) had a slope of ∼ 1, indicative of diffusive behavior. As the stiffness increased, the slope of the MSD increased, indicating more persistent behavior. The MSD was also larger for neutrophils on softer gels. (B) The angular distribution of steps is shown for each stiffness based on a 10-second interval between frames. The stiffer gels showed a more peaked distribution ∼ 0°, confirming that corresponding motion was more directed. Inset: The persistence, defined in the inset as the percentage of steps for which −π/2 < θ < π/2, is shown for time steps of 10, 20, and 30 seconds. The cells were more persistent on the stiffer gels. Error bars represent SE. *P < .05 vs 10 kPa.