Evos xl core inverted microscope

Transwell cell migration and invasion assays were carried out to assess the migration and invasion ability of U-251MG cells based on the previously described protocols [28 (link)]. Briefly, cells were transfected with pMEG3 or controls, incubated for 48 h, pelleted by centrifugation at 500 × g for 10 min at room temperature, and resuspended in serum-free medium RPMI1640 medium. 2.0 × 10⁴ cells were inoculated into the upper chambers of Transwell inserts (8 μm pore size; BD Bioscience, USA). The low chambers of the Transwell contained RPMI1640 medium with 10% FBS. The insert permeable membranes were either coated or not coated with Matrigel (Corning Life Sciences, USA) for assessment of cell invasion and migration, respectively. After 24 h incubation at 37°C, the cells remaining on the upper membranes were removed with a cotton wool, whereas the cells that had migrated or invaded through the membrane were stained with 2% crystal violet in 25% methanol/PBS, imaged, and counted in five randomly selected fields using an EVOS XL Core inverted microscope (Life Technologies, USA). The experiments were independently repeated three times.

For the assessment of invasion, transfected cells were cultured for 24 h and 2 × 10⁴ transfected cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (BD Bioscience, USA). Media containing 20% FBS were added to the lower chambers. After 24 h of incubation, the cells remaining on the upper membrane were removed with cotton wool, whereas the cells that had invaded through the membrane were stained with 2% crystal violet in 25% methanol/PBS for 20 min, washed three times with PBS, imaged and counted in five fields using an EVOS XL Core inverted microscope (Life Technologies, USA). The experiments were independently repeated three times.