Transwell Assay for Cell Invasion

Transwell assays were used to assess cell invasive capacity. Cell invasion assays were carried out using a BioCoat Matrigel Transwell chamber (BD, Franklin Lakes, NJ, USA) with a pore size of 8.0 μm. The inserts were placed in 24-well plates containing 700 μL of DMEM F12 1:1 medium for 30 minutes in a humidified 37 °C incubator under 5% CO₂ before seeding the cells. GANT61 (30 umol/L) was used to block the SHH signaling pathway, and after 48 hours, 5 × 10⁴ cells in each group resuspended in DMEM F12 1:1 medium containing 5% FBS were placed in each chamber. The lower compartment was loaded with full media containing 15% FBS as the nutritional attractant. After incubated at 37 °C for 48 hours, noninvaded cells were scraped off with a cotton swab. The translocated cells on the bottom of the upper chamber membrane were fixed with 5% formaldehyde and stained with 1% Giemsa stain. The number of cells that penetrated the upper compartment of the Transwell chamber was determined under an inverted microscope. Five fields of fixed cells were randomly chosen and counted under a light microscope [19 (link)].