Comparison of neutrophil spreading on substrates of different stiffness. (A) DIC images show neutrophils spreading over a period of 100 seconds on 4 gels of different stiffness (5, 10, 20, and 50 kPa), each at 3 time points. (B) The average spread area for each stiffness condition is plotted over time. Neutrophils on the softer gels maintained a smaller area and spread significantly less than the neutrophils on the stiffer gels. The majority of spreading was completed within the first 120 seconds. Neutrophils on the softest substrate showed no statistical change in size, even after 10 minutes, whereas those on the stiffest substrate more than tripled in area in the same amount of time. The number of cells measured for each condition is indicated in the legend. Error bars represent SE measurements. *Significant increase for the 10-kPa gels (with respect to the t = 0 point). #Significant increase for the 20-kPa gels (with respect to the t = 0 point). **Significant increase for the 50-kPa gels (with respect to the t = 0 point). Inset: The areas of the 4 neutrophils shown in the DIC images are plotted for the full 10 minutes.