LY294002 inhibits the ability of the neutrophil to sense stiff substrates. The root mean squared speed was calculated for neutrophils chemotaxing toward a pipette tip loaded with fMLP. Untreated cells and cells treated with 0.02% DMSO (vehicle control) and 20 μM LY294002 were recorded for a period of 30 minutes. No statistical difference was seen between the untreated cells and vehicle (DMSO)–treated cells. *Both the untreated and vehicle (DMSO)–treated cells showed a statistical difference between 10- and 100-kPa gels. Cells treated with LY294002 were indistinguishable on the 10- and 100-kPa gels. **The cells treated with LY294002 on the 100-kPa gels were significantly different from the untreated and vehicle cells on 100-kPa gels. In addition, cells treated with LY294002 showed limited ability to spread as shown in the representative DIC images below the plot. The DIC images are snapshots of single cells taken from supplemental Videos 3 through 6, respectively.