Cancer Cell Migration and Invasion Assay

Endpoint migration and invasion assays were performed using a transwell system in 24-well plates (353504, Falcon). FluoroBlock™ filter inserts (351152, Corning) with 8 μm pore size were used that block light transmission from 400-700 nm and allow fluorescence detection only from the cells that migrated/invaded to the bottom side of the inserts with a bottom-reading fluorescence plate reader. The inserts were uncoated for the migration assays, and coated for the invasion assays with 0.2 mg/ml type I bovine collagen PureCol^® of which the pH was neutralized to 7.2 – 7.4 using sodium bicarbonate. Cancer cells that were serum starved for 24 hrs were seeded onto the inserts at 0.5x10⁵ cells/ml in 100 µl of serum free media. Cells were allowed to migrate toward the bottom chamber containing 500 µl of full-serum media as the chemoattractant or serum-free media as the negative control. After a 24 hr incubation at 37°C/5% CO₂, the inserts were transferred to a fresh 24-well plate with black walls containing 500 μl of 4 μM Calcein AM (Biotium) in mHBSS per well. Cells were incubated for 1 hr at 37°C, and the fluorescence reading of migrated/invaded cells was measured from the bottom at wavelengths of 495/515 nm (Excitation/Emission) by a SpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices).