Transwell cell culture chambers with 8 μm pores

Cell migration and invasion were analyzed using Transwell cell culture chambers with 8-μm pores (Corning, USA). The upper chamber was coated with Matrigel (Corning) for invasion assays, but not for migration assays. Cells (10⁵) were added to the upper chamber without FBS, while lower chambers were immersed in medium containing 10 % FBS as a chemoattractant; the culture chambers were then incubated for 24 h at 37 °C. Cells were removed from the upper chamber using cotton swabs, and the filters were stained with methanol for 5 min at 37 °C, then fixed with Giemsa’s solution (Solarbio, China) for 10 min at 37 °C. Numbers of migrating and invasive cells were counted in five randomly selected fields on each filter. Representative data from three independent experiments are shown.

In vitro cell invasion assays were performed using Transwell® cell culture chambers with 8 μm pores (Corning, NY, USA). The inserts in the membrane filter were coated with 40ul configured Matrigel™ on the upper surface. The cells were resuspended in serum-free DMEM at a concentration of 5 × 10⁵ cells/ml and placed in the upper chamber. The lower chamber was filled with DMEM with 10% FBS. After incubation at 37°C for 48 hours, the cells on the upper surface of the filter were removed with a cotton swab. The invading cells at the bottom of the Matrigel™ were fixed in methanol and stained with 0.1% crystal violet. The number of invading cells in five random fields per well was calculated using a microscope at 200× magnification. Each assay was performed in triplicate.