2.10 |. ELISA-based quantification of small GTPase activity

Commercial ELISA kits were purchased from Cytoskeleton (G-LISA) to quantify RhoA and Rac1 activation (Cytoskeleton). For both cases, cell lysates were prepared using Cytoskeleton’s protocols. Following exposure to flow or static conditions, the collagen/hyaluronan hydrogel was removed from the device and washed in ice-cold 1× PBS for 30 s. 50 μl of ice-cold cell lysis buffer with 1× protease inhibitor were injected into vessels and collected in a microcentrifuge tube. The cell lysate solution was spun at 10 000 g for 1 min at 4°C to pellet cell debris. The supernatant was collected and snap-frozen in liquid nitrogen, reserving a small amount for protein quantification using Precision Red (Cytoskeleton). Prior to measuring GTPase activity, samples were thawed in a room temperature water bath and equilibrated to 0.5–1 mg/ml for RhoA and 0.5 mg/ml for Rac1 samples. Sample numbers of n≥3 were used to determine the mean and standard deviation of GTPase activation for each condition.