Ckx31 inverted light microscope

Transwell chambers (insert diameter, 6.5 mm; pore size, 8.0 µm; 24 wells) were coated with 30 µl Matrigel (BD Biosciences) for 30 min at 37°C. DMEM (600 µl) containing 10% FBS was added to the lower chamber. A total of 100 µl cell suspension (1×10⁶ cells/ml) in serum-free medium was added to the upper Transwell chamber. After 24 h of incubation at 37°C, U251 cells that had invaded across the membrane were stained with 0.1% crystal violet for 20 min at room temperature. Non-invasive cells in the upper chamber were gently wiped off with a cotton swab and washed 3 times with PBS. The numbers of invasive cells were observed in five field power magnification under an Olympus CKX31 inverted light microscope (Olympus Corporation, Tokyo, Japan; magnification, ×400) and counted. The procedure was repeated for U87 cells.

For the cell invasion assay, 3×10⁴ cells were seeded into the upper chamber of Transwell assay inserts (8 µm; Corning Inc.). The Transwell chamber was precoated with Matrigel (Thermo Fisher Scientific, Inc.) before cell seeding. The chambers were inserted into a 24-well plate. The upper chambers were filled with 200 µl serum-free medium (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.), while the bottom chambers were filled with 500 µl complete medium (RPMI-1640; Gibco; Thermo Fisher Scientific, Inc.). After incubation at 37°C for 24 h, the cells were fixed at room temperature in 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 10 min and stained at room temperature with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 10 min. Nonmigrating cells in the upper chambers were wiped off. The migrated cells were counted in three randomly selected fields and photographed at a ×100 magnification with a CKX31 inverted light microscope at ×100 magnification (Olympus Corporation).