To prepare the gel layers, the components of the HRI gel pre-polymers, parts A and B of QGel 920 and parts A and B of QGel 903 (both by Quantum Silicones LLC, Richmond VA; refractive index of 1.49 when cured), were mixed in various proportions (Table 1 ) and coated onto 25 mm no. 1 round cover glasses using a home-built spin-coater rotating at 1920 rpm. Each cover glass was baked at 100°C for 2 hr to create a layer of cured gel on it with a thickness, µm. After baking, the gels on the cover glasses were treated with 3-aminopropyl trimethoxysilane for 5 minutes and incubated for 10 minutes at room temperature under a suspension of 40 nm carboxylated far-red fluorescent beads (excitation/emission 690/720 nm, by Invitrogen, Carlsbad, CA) in a 100 µg/ml solution of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in water to covalently link beads to the gel surface. This technique made it possible to have all beads in one plane corresponding to the surface of the gel. Therefore, the beads could be imaged under wide-field (epi-fluorescence illumination) with minimal background and their displacements reflected the deformation of the very top of the substrate. To promote cell adhesion, fibronectin (FN) was covalently linked to the gel surface by incubation in 50 µg/ml of FN with 100 µg/ml EDC in PBS, pH 7.4 for 30 min at room temperature.
The elastic modulus (Young's modulus), E, of the gels was evaluated by applying a known hydrodynamic shear stress, τ, to the gel surface using a custom-built microfluidic device, measuring the resulting bead displacement, , calculating the shear of the gel, , and applying the equation , where ν is the Poisson ratio, as explained in detail elsewhere [26] . Because the Poisson ratio of silicone gels is nearly equal to 0.5 [27] , the equation was reduced to . To measure the gel thickness, , a small amount of the 40 nm far-red fluorescent beads was deposited on the cover glass surface before it was coated with the gel pre-polymer. The fluorescence microscope was first focused on beads on the glass surface and then on those on the gel surface and the difference in the readings of the nosepiece (z-axis) knob was recorded (with a correction for the mismatch between the refractive indices of the gel and immersion liquid), resulting in ∼1 µm accuracy. The shear, , was found to be a zero-crossing linear function of τ for of up to at least 3 µm (greater than produced by HUVECs; see below) for all gels, with no sign of plastic deformations (see also [26] ), thus validating the use of the equation , which applies to linear materials. Furthermore, measurements of vs. at different constant values of τ resulted in linear dependencies, indicating homogeneity of mechanical properties of the gel layers.
The elastic modulus (Young's modulus), E, of the gels was evaluated by applying a known hydrodynamic shear stress, τ, to the gel surface using a custom-built microfluidic device, measuring the resulting bead displacement, , calculating the shear of the gel, , and applying the equation , where ν is the Poisson ratio, as explained in detail elsewhere [26] . Because the Poisson ratio of silicone gels is nearly equal to 0.5 [27] , the equation was reduced to . To measure the gel thickness, , a small amount of the 40 nm far-red fluorescent beads was deposited on the cover glass surface before it was coated with the gel pre-polymer. The fluorescence microscope was first focused on beads on the glass surface and then on those on the gel surface and the difference in the readings of the nosepiece (z-axis) knob was recorded (with a correction for the mismatch between the refractive indices of the gel and immersion liquid), resulting in ∼1 µm accuracy. The shear, , was found to be a zero-crossing linear function of τ for of up to at least 3 µm (greater than produced by HUVECs; see below) for all gels, with no sign of plastic deformations (see also [26] ), thus validating the use of the equation , which applies to linear materials. Furthermore, measurements of vs. at different constant values of τ resulted in linear dependencies, indicating homogeneity of mechanical properties of the gel layers.
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