Cell migratory ability was evaluated with the use of the Transwell polycarbonate membrane inserts (Millipore, Billerica, MA, USA). The transfected CAL27 and SCC-4 cells (1 × 105) were planted into the upper chamber of the insert. 10% FBS was used to supplement cell-free medium in the low chamber at 37 °C for 1 day. Cells migrating through the membranes were cultured in 4% paraformaldehyde and stained with 0.1% crystal violet. For cell invasion assay, 1 × 105 cells in sterile medium were seeded into the upper chamber with Matrigel (Sigma-Aldrich, St. Louis, MO, USA). Both migrated and invaded cells were observed and counted under an inverted light microscope (Leica Microsystems, Wetzlar, Germany) at a magnification of ×200.
Transwell polycarbonate membrane inserts
Manufactured by Merck Group
Sourced in Germany
Transwell polycarbonate membrane inserts are a laboratory equipment product designed for cell culture applications. The inserts feature a porous polycarbonate membrane that allows for the separation and co-culture of different cell types within a single well. The membrane's pore size and density can be customized to suit specific experimental requirements.
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4 protocols using transwell polycarbonate membrane inserts
1
Cell Migration and Invasion Assay
2
Transwell Assay for Cell Migration
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Cell migration ability assessment was performed using Transwell polycarbonate membrane inserts (Millipore, Schwalbach, Germany). The cells (1×105) were plated onto an insert and 20% FBS was added to the cell-free medium in the lower chamber. After incubation 24 h at 37°C, the inserts were washed in PBS, and the cells were fixed to the membranes with 4% paraformaldehyde, then stained with Hoechst (10 μg/ml). The migrated cells were counted per high-power field.
3
TUFT1 siRNA Impacts Cell Invasion
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Transwell assays were performed to examine the invasion of cells transfected with small interfering RNA that targeted TUFT1 or a scrambled negative control. Cell invasion was defined using Transwell polycarbonate membrane inserts (Millipore). Cells (1 × 105) in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (TheWell Bioscience Inc., North Brunswick, NJ, United States). Medium containing 10% FBS was added to the lower chamber to stimulate invasion, and the cells remaining on the upper membrane were removed with cotton wool after cultivation for 48-72 h. Cells that had invaded through the membrane were stained with methanol and 0.1% crystal violet, imaged, and counted using an IX71 inverted microscope (Olympus, Tokyo, Japan). Each assay was repeatedly conducted three times.
4
Cell Migration Assay Using Transwell
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The cell migration experiment was performed using Transwell polycarbonate membrane inserts (Millipore, Schwalbach, Germany) on 24-well plates. The cells (5 x 10 4 ) were plated onto the chamber and vascular endothelial growth factor (VEGF) (50 ng/mL) was added to the cellfree medium in the lower chamber. After incubation for 24 h at 37°C, the inserts were rinsed in PBS, and the cells were fixed to the membranes with 4% paraformaldehyde and stained with Hoechst (10 mg/mL). The migrated cells were counted at high-power field.
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