Neutrophil transmigration on stiff substrates causes injury to the monolayer. Shown are representative phase-contrast images of the HUVEC monolayer after neutrophil transmigration on (A) a soft (0.87-kPa) substrate and (B-C) stiff (280-kPa) substrates. Time after plating neutrophils onto the HUVEC monolayers (T) is shown at the top of each image. Large holes commonly form in monolayers on stiff substrates after transmigration and are outlined in panels B and C by white dotted lines. (D) Also shown is a phase-contrast image of a monolayer on a 280-kPa substrate at approximately 2 hours after plating neutrophils. Significant neutrophil accumulation in the area of the hole has occurred. Scale bar in panel D is 50 μm and applies to images in panels A-D. (E) Shown is a time sequence of a hole forming and then healing on a 5-kPa substrate. Time after plating neutrophils is indicated at the bottom of each image. The scale bars on all images in panel E are 20 μm.