Dp controller and dp manager software

2 × 10⁴ cells were seeded in medium with 0.05% FBS in the inner chamber of Costar Transwell cell culture inserts (6.5-mm diameter, 8-μm pore size; Corning, Corning, NY). Medium with 5% FBS was added to the outer chamber as chemoattractant. Cells were incubated for 22 h, and then chambers were immersed in 0.5% crystal violet and 25% methanol for 10 min. Images of migrating cells were acquired with the Olympus DP71 fluorescent microscope and DP Controller and DP Manager software (Olympus). The number of migrating cells per field of view in each condition was quantified using ImageJ software to analyze 6 separate fields of view each of the 3 replicate wells.

1 × 10⁵ cells were seeded on poly-L-lysine- (Merck) coated coverslips in 24-well plates. 48 h after seeding, cells were fixed with 4% formaldehyde in Dulbecco’s PBS (DPBS) for 20 min at room temperature. Cells were blocked with 5% normal goat serum (Invitrogen, Carlsbad, CA) and 0.3% Triton X-100 (Sigma-Aldrich) in DPBS for 1 h at room temperature, then incubated with primary antibodies in diluent buffer (1% BSA and 0.3% Triton X-100 in DPBS) overnight at 4°C. The next day, cells were washed and incubated with secondary antibody in diluent buffer for 2 h at room temperature, protected from light. Coverslips were mounted on SuperFrost Ultra Plus glass slides (Menzel-Gläser, Thermo Fisher Scientific, Waltham, MA), using SlowFade Diamond Antifade Mountant (Molecular Probes, Eugene, OR). Images were acquired with the Olympus DP71 fluorescent microscope and DP Controller and DP Manager software (Olympus, Shinjuku, Japan). Images were quantified using ImageJ software. Antibodies used for immunofluorescence are listed in Table S7.