Biocoat matrigel invasion kit

2.5×10⁴ serum-starved cells were added to the chamber of BioCoat Matrigel Invasion Kit (BD Biosciences, Franklin Lakes, NJ, USA, #354480) with complete culture media containing 10% FBS at the bottom. After being incubated for 24 hours, non-invading cells were removed from upper surface of the membrane using cotton-tipped swabs. Blind counts of invaded cells were obtained following Crystal Violet staining of live cells. This assay was performed with four replicates for each cell type.

Cell migration and invasion experiments were performed using the Transwell chamber (BD Science, Bedford, MA, USA) according to the manufacturer’s instructions. The 143B cells were cultured in the serum-depleted McCoy’s 5A medium and cultured for 24 h. Then, the cells were trypsinized and resuspended in serum-depleted McCoy’s 5A medium. The 5×10⁴ cells were placed in the apical chamber of the Transwell plate and the 90% McCoy 5A medium containing 10% FBS was placed in the basolateral chamber and incubated for 24 h. The remaining cells in the apical chamber were removed with a sterile cotton swab, the migrated cells were fixed with 4% formaldehyde on the lower surface of the filter, stained with 2% crystal violet, counted, and imaged under an optical microscope. Cell invasion assay was similar to cell migration assay except that the filter membrane of Transwell apical chamber was coated with Matrigel in BioCoat Matrigel invasion kit (BD science). The cells were counted and imaged using an optical microscope.