Biocoat matrigel

To determine the effect of PBMC on the invasiveness of breast cancer cell lines (MCF-7 and MDA-MB-231), cell invasion assays were performed in BD BioCoatMatrigel invasion chambers (24-well plates, Corning, Biocoat Matrigel™, 354480). Briefly, the breast cancer cell lines (MCF-7: 5 × 10⁴ cells; MDA-MB-231: 1 × 10⁴ cells) were seeded in DMEM-F12 in the upper chamber, whereas PBMC were seeded at 2 × 10⁶ cells/mL in DMEM-F12 in the lower chamber. DMEM-F12 without PBMC was used as control. After incubation for 48 h, cells that migrated to the lower surfaces of the filters were fixed in cold absolute methanol for 20 min, stained using a 0.5% crystal violet solution and 20% methanol for 30 min in the dark, visualized, and counted. Values for cell invasion were expressed as the mean number of cells per microscopic field over ten fields.

Cell migration assay was carried out using Transwell (Corning Costar Corp., Cambridge, MA, USA) membrane filter inserts in 24-well tissue culture plates with 6.5 mm diameter and 8μm pore size. Following CD44 overexpression or knockdown, lung cancer cells were trypsinized and suspended in serum-free medium. The cells were then seeded on the upper chamber of Transwell, and FBS-containing medium was added to the lower chamber. After 24 h of incubation at 37 °C, crystal violet was used to stain the cells. Non-migrated cells from the upper chamber were removed, and migrated cells were imaged by Oympus100 and analyzed by using Image J software. For invasion assay, BioCoat Matrigel (Corning, Bedford, MA, USA) Transwell inserts were used, and the procedure was similar to Transwell migration assay and according to the manufacturer’s instructions.

The cell invasion detection was conducted as described previously (Orucevic et al., 1999 (link)). Transwell chambers (Corning^® BioCoat^™ Matrigel^®, USA) were used in the cell invasion detection method. The experiment was designed according to the following principle, which demonstrated that cancer cells (as HT29 cells) tended to migrate into the outer chambers containing 10% FBS. HT29 cells were cultured (1 × 10⁴ cells/well) in 150 μL serum-free medium, and LF (6.25 μM), OA (0.18 mM), DHA (0.18 mM), LA (0.15 mM), 5-Fu (2.0 μM), LF + OA, LF + DHA, or LF + LA was added to the insert respectively. Meanwhile 500 μL RPMI-1640 complete medium (supplemented with 10% FBS) were added in the outer chambers. Cells were cultured for 24 h, and cotton swabs were used to scrub the filters surfaces. Migrated cells were treated with ice-cold methanol for 15 min, while the undersides were dyed by 0.2% crystal followed by washing with ice-cold PBS (3 min × 5). The dyed cells on the undersurfaces were counted and calculated in three random fields.