Biocoat control inserts with 8.0 μm pet membrane

U138 glioma cells and SK1035 GSCs were transfected with control siRNA or siRNA against IMP3. After 36 h of transfection, cells were plated for p65 overexpression transfection (5 μg in a 35 mm). After 36 h of p65 transfection, cells were made into single cell suspension and 25,000 cells were plated for migration in a Corning^® BioCoat^™ Control Inserts with 8.0 μm PET Membrane (Catalog # 354578, Corning, U.S.A.). Upper chamber contained incomplete medium required for the growth of respective cell lines, while the lower chamber contained medium with 20% serum. Migration of cells was observed after 8 h of incubation in 37^°C incubator. Cells from the upper side of the chamber were gently removed, while those in the lower side were fixed using chilled methanol (30 min) and stained with 0.1% crystal violet (30 min). Representative images were taken under light microscope and migrated cells were counted. The assay was performed in duplicates.