| 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 alFibronectin 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: - Table S1. Reported values of tissue stiffness (PDF, 77.1 KB) -
Values of stiffness measurements for a representative set of tissue types are listed with the original publication and the technique used.
- Figure S1. Fibronectin coating is unaffected by the stiffness of the gels (JPG, 24.6 KB) -
Gels were prepared and coated with fibronectin as described in the Methods section. Fluorescent beads 1 µm in diameter were coated with anti-fibronectin antibody and incubated with the gels. After vigorous washing, images of the surface of each gel were taken, and the number of beads per field of view calculated. Three images were taken for three different gels of each stiffness. No difference was seen in the number of beads on the gels of different stiffness. This confirms that the protein coating on the gels is uniform and density is independent of substrate stiffness.
- Video 1. A neutrophil migrating on a polyacrylamide gel (MOV, 103 KB) -
This video corresponds to the DIC images shown in Fig. 6A. The total elapsed time in the video is 350 s.
- Video 2. The calculated traction map of neutrophil migrating on a polyacrylamide gel (MOV, 128 KB) -
This video corresponds to the traction maps calculated for the neutrophil shown in Fig. 6A and Video 1. The total elapsed time in the video is 350 s.
- Video 3. Neutrophils migrating towards a micropipette tip loaded with fMLP on a 10 kPa polyacrylamide gel (MOV, 4.46 MB) -
The neutrophils do not spread out as they migrate towards the pipette tip. The total elapsed time is 20 min.
- Video 4. Neutrophils migrating towards a micropipette tip loaded with fMLP on a 100 kPa polyacrylamide gel (MOV, 2.85 MB) -
Neutrophils flatten and spread out, tripling their size and show a distinctive ruffling at their leading edge as they migrate towards the pipette tip. The total elapsed time is 20 min.
- Video 5. Neutrophils pretreated with 20 µM LY294002 migrating towards a micropipette tip loaded with fMLP on a 10 kPa polyacrylamide gel (MOV, 3.66 MB) -
Neutrophils pretreated with the PI3K inhibitor behave similary to untreated neutrophils on a 10 kPa gel. The total elapsed time is 20 min.
- Video 6. Neutrophils pretreated with 20 µM LY294002 migrating towards a micropipette tip loaded with fMLP on a 100 kPa polyacrylamide gel (MOV, 3.83 MB) -
Neutrophils no longer spread out like the untreated cells on the 100 kPa gel, and are indistinguishable from the neutrophils on the 10 kPa gel. The total elapsed time is 20 min.
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