Collagen coated glass bottomed dishes

Collagen-coated glass-bottomed dishes (MatTek, Ashland, MA) were coated with 0.2% Matrigel in serum-free medium for 30 min at 37°C. Cells were seeded sparsely overnight and then serum starved for 4 h in Leibowitz’s L-15 medium (Life Technologies) with 0.35% BSA. Differential interference contrast images were acquired every 20 s for 10 min in an environmentally controlled microscope (TE2000; Nikon) with a 20× objective and a Photometrics CoolSNAP HQ camera. Growth factor/inhibitor solutions were added after 80 s. Cell areas were traced immediately before and 9 min after stimulation using ImageJ (National Institutes of Health, Bethesda, MD). Data shown are from individual cells (at least 60 overall) pooled from at least three separate experiments.

Cells were plated in a haptotaxis device on a 125 μg/ml 2D FN gradient for 3 h or on collagen-coated glass-bottomed dishes (MatTek) in serum-free medium for 30 min at 37°C. Cells were then fixed for 20 min in 4% paraformaldehyde in PHEM buffer (60 mM piperazine-N,N′-bis(ethanesulfonic acid), pH 7.0, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.0, 10 mM ethylene glycol tetraacetic acid, pH 8.0, 2 mM MgCl₂, and 0.12 M sucrose) and then permeabilized with 0.2% Triton X-100, blocked with 10% BSA, and incubated with primary antibodies overnight at 37°C. Z-series of images were taken on an Applied Precision DeltaVision microscope using Softworx acquisition, an Olympus 40×/1.3 NA Plan Apo objective, and a Photometrics CoolSNAP HQ camera. Images were deconvolved using DeltaVision Softworx software and objective-specific point spread function. Images were analyzed with ImageJ. Images are pooled from at least three independent experiments, with at least 10 cells per experiment.