EC hole formation begins when neutrophil transmigration has reached a maximum. (A) Fraction of neutrophils that have transmigrated (primary vertical axis) as a function of time after addition of neutrophils is shown for varying substrates (0.87, 5, 280 kPa). Also plotted is the number of holes per area (secondary vertical axis) as a function of time after addition of neutrophils, for varying substrates and number of neutrophils plated. (B) Data from panel A at T = 45 minutes are highlighted. Shown is the number of holes per area on each of the substrates, with varying numbers of neutrophils. (C) The area of holes at T = 45 minutes is quantified for varying substrate stiffness and number of neutrophils plated. Bars represent average. Error bars represent SE. *P < .05 (using ANOVA). ***P < .001 (using ANOVA).