Cls3422

Before migration, cells were serum-starved overnight. Then, 2 × 10⁵ OS cells or 1 × 10⁶ HUVECs in 200 μl of the serum-free RPMI 1640 medium were seeded to each top well of a 12-well Transwell Boyden system (8 μm pore size, Sigma, CLS3422), and 500 μl RPMI 1640 medium supplemented with 10% fetal calf serum was added to the lower chamber. Cells were allowed to migrate for 24 h at 37 °C, in 5% CO₂. After removing cells on the upper surface of the filter using cotton swabs, cells that invaded through the membrane were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 15–20 min. The number of cells that reached the lower part of the Transwell filter membrane was counted with Image J software (National Institutes of Health, USA) and plotted as the number of cells per optic field (× 200). Experiments were carried out in triplicate.
The invasive procedure is almost the same as the migration assay except for the 8 μm pore size (corning, CLS3422) coated with 150 μg of Matrigel (65 μl of 2.3 mg/ml of Matrigel, Becton Dickinson Matrigel Basement membrane Matrix, phenol-red free, Collaborative Research Cat. no. 40234C) used.

Transwell permeable supports that have 6.5 mm diameter inserts of polycarbonate membrane with 8-µm-diameter pore size (Corning, CLS3422) were used in the experiments reported here. Each Transwell insert was coated with 80 μL of 10 µg mL⁻¹ fibronectin solution (in 1× PBS, Corning Inc., 354008) and left to dry for 12 h. Cells were simultaneously serum starved in migration media for 12 h. Following this step, the cells were removed using 0.05% trypsin-0.02% EDTA solution (Sigma-Aldrich, 59417 C) and handled exclusively in migration media. Cell suspensions with a concentration of 1 × 10⁶ cells mL⁻¹ were prepared and 150 µL of this media (1.5 × 10⁵ cells) was plated in the top chamber of each Transwell insert and the bottom chamber was filled with 600 µL of migration media or EGF supplemented migration media (100 ng mL⁻¹ EGF). After 8 h, the cells were fixed using a standard HEMA 3 solution kit (Fisher Scientific, 23-123869) and imaged using a Stereo Microscope (Leica Microsystems Inc.). Cell migration numbers were then quantified using a custom MATLAB script as described in Image Acquisition and Processing. Cell migration without growth factors and without iEFs served as controls and all the other conditions were normalized with respect to these controls.

Wound healing and transwell assays were used to assess cell migratory ability. The wound healing experiment was performed by using a wound healing assay kit (Abcam, ab242285) according to the manufacturer’s instructions. The procedures for transwell assay (Corning, Corning, NY, USA, CLS3422) were conducted as previously described [14 (link)].

Cell migration and invasion abilities were assayed with a transwell system containing 8.0-µm pores (Corning, USA, CLS3422). Cells in the logarithmic growth phase were synchronized by serum starvation overnight. For cell invasion assays, transwell inserts were coated with 50 µl of a mixture of serum-free RPMI 1640 and Matrigel (1:8). Matrigel was allowed to solidify at 37℃ for 4 h. Synchronized cells in 100 µl serum-free RPMI 1640 were seeded on the upper chamber and allowed to settle for 20 min, and 600 µl of complete RPMI 1640 medium was added to the lower chamber. For the cell migration assay, synchronized cells were seeded on the upper chamber. Cells were incubated in a humidified incubator with 5% CO2 for 24 h at 37℃. The cells that migrated to the lower chamber were washed with PBS three times, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet at room temperature for 30 min. The number of cells that migrated through the pores or invaded the Matrigel was counted. Cells in five representative fields were counted in each membrane.

6.5 mm diameter Transwell inserts (Corning, CLS3422) coated in 1.4% gelatin were seeded with primary human umbilical vein endothelial cells (HUVEC) (Promocell C-12200, kindly provided by Dan Gioeli at UVA) at a density of 1500 cells/mm² (50,000 cells/insert). Cells were cultured in complete endothelial cell growth media (Promocell, C-22110) and incubated at 37°C and 5% CO₂. After 24 hours, the cells were washed in sterile PBS and the media was changed.