Matrigel coated membrane 24 well insert

Cell migration and invasion was measured in a Boyden chamber system as described [11 (link)]. For migration assays, cells (1 × 10⁵) were placed in the upper chamber with non-coated membrane (24-well insert; 8-μm pore size; Corning Inc., Corning, NY). For invasion assays, cells (1 × 10⁵) were placed in the top chamber with Matrigel-coated membrane (24-well insert; 8-μm pore size; Corning Inc.). The total number of cells that migrated into the lower chamber was counted after 16 h of incubation at 37 °C with 5% CO₂. Cells that migrated to the lower surface of the filter were stained with 0.5% crystal violet, examined by bright field microscopy, and photographed. Crystal violet was then dissolved with ethanol, and absorbance was read at 570 nm. Values for migration/invasion are expressed as the average number of OD570 per assay.

For transwell migration assay, these differently treated MDA-MB-231 cells (2.5 × 10⁴, for 4 h) were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 μm; Corning). For invasion assay, MDA-MB-231 cells (5 × 10⁴, for 4 h) were plated in the top chamber with the Matrigel-coated membrane (24-well insert; pore size, 8 μm; Corning). In both assays, MDA-MB-231 cells were re-suspended in DMEM without serum, and the 20% serum-supplemented DMEM was used as a chemoattractant in the lower chamber. These cells were incubated under hypoxic environment for 24 h, and then the residual non-migrated or non-invaded cells were removed by a cotton swab. After staining with CV, the lower membrane surface-fixed cells were counted and imaged through inverted microscope.

For migration assays, 6×10⁴ cells were plated in chambers with the non-coated membrane (24-well insert; pore size, 8μm; Corning). For invasion assays, 8×10⁴ cells were plated in chambers with Matrigel-coated membrane (24-well insert; pore size, 8μm; Corning). After a 24h incubation, cells on the upper surfaces of the membrane were removed by a cotton swab and cells on the lower surfaces of the membrane were stained with the crystal violet and counted using an inverted phase-contrast microscope.