8 μm cell culture insert

Cells were trypsinized, counted, resuspended in medium containing 0.5% serum and then transferred to 8-μm cell culture inserts (BD Falcon) placed into 24-well plates containing complete medium. After 16 h, cells were removed from the top of the filter using a cotton swab, and migrating cells on the bottom of the filter were fixed with 4% PFA and stained with 5% crystal violet^{26 (link)}.

Serum starved treated cells were seeded into 8 μm cell culture inserts (Falcon, #Cat 353097) in 24-well culture plates (Falcon). Media with 5% FBS was added to the lower chamber as a chemoattractant. Assay plates were transferred to a 37°C incubator and let undisturbed for 24 h. Cells that had invaded the surface of the filter were fixed with Formalin 10%, (Hemochem) and stained with Crystal Violet 0.5% in 25% Methanol, (BDH). Inserts with stained cells were imaged at HMR imaging facility and quantified using ImageJ.

Cell migration assay was performed using an 8-μm cell culture insert (Falcon). Cell invasion was examined using polycarbonate membrane Transwell inserts (Costar; Corning Inc.). After 48 h of transfection, cells were added to the upper chamber (2 × 10⁴ cells/well) with 200 µl serum-free medium. The upper chamber was incubated for 24 h in a 24-well plate chamber with 200 µl complete medium containing 10% FBS. After the cells at the top of the upper cavity were wiped with a cotton swab, the transplanted cells at the bottom of the cavity were stained in 0.1% crystal violet solution at room temperature for 10 min. The migrated and invading cells were photographed under an inverted microscope and counted in four random fields.