Blood, Vol. 114, Issue 7, 1387-1395, August 13, 2009

Neutrophil morphology and migration are affected by substrate elasticity
Blood Oakes et al. 114: 1387

Supplemental materials for: Oakes et al

Fibronectin coating is not affected by substrate stiffness
The uniformity of the FN coating on the acrylamide substrate was measured by immunofluorescence, similar to the procedure described by Lo et al.1 Acrylamide gels were made and coated with FN as described in the Methods section, before being incubated with a 100 µg/mL solution of mouse anti-human FN monoclonal antibody (Sigma-Aldrich, St. Louis, MO) for 1 hr at room temperature. The substrate was then washed three times with PBS. Fluorescent beads (Invitrogen, Carlsbad, California), 1 µm in diameter, were washed in PBS and resuspended in 22 µg/mL solution of Protein A/G (Pierce, Rockford, IL) overnight at 4°C. The beads were washed and resuspended in 20 µg/mL goat anti-mouse IgG (Zymed Laboratories, South San Francisco, CA) and placed in a waterbath at 37°C for 1 hr. The beads were again washed in PBS and incubated with the gels for 1 hr at room temperature. After incubation the gels were vigorously washed with PBS and examined under the microscope. The images were thresholded and the number of beads counted per field of view (Fig. S1).

1. Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the rigidity of the substrate. Biophys J. 2000;79:144-152.

Files in this Data Supplement: