Biocoat matrigel chamber

Migration or invasion assays were performed by plating cells in Transwells (Corning, 6.5 mm, 8.0 mm pore size) or BioCoat Matrigel chambers (BD Biosciences, 6.5 mm, 8.0 mm pore size), respectively, in serum‐free medium with 0.01% bovine serum albumin. Chemotactic migration/invasion was induced be adding 10% FCS to the lower chamber. For MDA‐MB‐231 and mesHMLE cells, 2 × 10⁵ or 8 × 10⁴ cells were utilised in 4‐h migration or 24‐h invasion assays, respectively. For SH‐EP cells, 8 × 10⁴ cells were utilised in 24‐h invasion experiments. Following experimental endpoint, membranes were fixed with 10% buffered formalin and cells stained with DAPI. Six fields of view for each membrane were counted using FIJI software (Schindelin et al, 2012). All experiments included at least 3–4 replicates and were performed multiple times with data pooled and shown as mean ± SEM.
For non‐directional cell migration, transfected cells were plated at low density and images were collected on the IncuCyte Zoom (Essen Bioscience) every hour for 24 h. Cell tracking was performed manually with 25 cells per group using the manual tracking and chemotaxis tools on FIJI software (Schindelin et al, 2012).

Invasion assay were performed in BD BioCoat Matrigel Chambers. 50,000 cells/well were plated in the upper chamber whereas SDF-1α (100 ng/ml) or 10% FCS were added to the lower chamber in serum free medium. After 22 hours of incubation in a humidified atmosphere 5% CO₂ at 37°C, non invading cells were removed from the upper surface of the membrane and invading cells were fixed and stained with Diff-Quik (Medion Diagnostic) before counting.

Cell migration was assessed by using the 24-well transwell plate with a polycarbonate filter membrane of 8 μm pore size (Corning, Acton, MA, USA). The upper compartment of the transwell chamber was seeded with THP-1 cells at a density of 1×10⁵ in 200 μl of serum-free medium, and the lower chamber was seeded with Cal-27 cells at a density of 1×10⁴ in 500 μl of DMED medium. For the cell migration assay, after 12 h and 24 h incubation, the medium was discarded and the filter membrane was fixed with 4 % formaldehyde, stained with 0.1 % crystal violet and examined under an inverted microscope to count migrated cells in 5 different fields. For the cell invasion assay, macrophages were seeded to the upper compartment of BD BioCoat Matrigel chambers (BD Biosciences, San Jose, CA, USA) at a cell density of 1×10⁶ and incubated for 24 h and 48 h. After incubation, the invaded cells on the lower surface were stained with 0.1 % crystal violet and counted in 5 randomly selected fields. To detect the cell migration and invasion of Cal-27 cells, the same experiment was applied and the upper chamber was seeded with Cal-27 cells while the lower chamber was seeded with THP-1 cells.

Migration and invasion assays were performed by seeding 50,000 cells per well in BD BioCoat matrigel chambers (BD Biosciences). After 20 hours, invading or migrating cells were fixed and stained as per the manufacturer's instructions and counted at 20X magnification (EVOS cell imaging system, Life Technologies).