8 μm pore size transwell chamber

Cell migration assays were performed using 8 μm pore size transwell chambers (Corning, NY, USA). The lower chamber was filled with normal culture medium (DMEM with 10% FBS). Cells were suspended in DMEM with 1% FBS and seeded in the upper chamber. After 18 h, cells were stained with 0.5% crystal violet, and the number of cells on the bottom surface of the polycarbonate membrane was counted using an optical microscope. The same procedure was used for cell invasion assays, except that the upper chamber was filled with matrigel.

We established the coculture system through 6-well plate and 3 μm pore size transwell inserts (Corning). The SMCs (5 × 10⁵ per well) were seeded in 6-well plate and the same quantity of different treated ECs were seeded in the transwell inserts located in adjoining wells. The transwell inserts with ECs were moved to the wells containing SMCs when cells attached to the wall firmly (24 h). After another 48-h culture, SMCs were harvest for western blot. Similarly, we set up another co-culture system for migration experiments, 24-well plate and 8 μm pore size transwell chambers (Corning) were used instead, 5 × 10⁴ different treated ECs per well were seeded in 24-well plate and the same number of SMCs were seeded in up chambers adjacently. After cells attached firmly, the transwell chambers with ECs were moved to the wells containing ECs. Forty eight hours later, chambers were taken out for migration experiments.

For invasion assay, 8 μm pore size transwell chambers (Corning Incorporated) was pre‐coated with Matrigel matrix (BD Biosciences). Briefly, BCPAP and TPC1 cells in serum‐free medium were seeded into the upper chamber. The lower chamber was filled with complete medium. After 48 hours, cells in the down chamber were fixed and counted using a light microscope.

The cell migration and invasion experiments were performed using 8 μm pore size transwell chambers (Corning) in 24-well plates. Cell medium containing 10% FBS was added to 24-well plates 0.6 ml per well. The 200 μl serum-free medium containing certain number of cells were added into each upper chamber. For migration, 4–5 × 10⁴ cells were diluted in 200 μl serum-free medium and added to each chamber. For invasion, the chambers were pre-coated with 50 μl diluted Matrigel (Corning, Matrigel: medium = 1:8), and then 8–10 × 10⁴ cells were diluted in 200 μl serum-free medium and added to each chamber. After incubation at 37 °C for 24 h, cells on the upper surface of chamber were swept by cotton swab slightly. Cells on the bottom of the chamber were fixed with paraformaldehyde for 15 min and stained with crystal violet for 30 min. Cells penetrated were imaged and counted at five random fields.

Cell invasion was evaluated using 8-μm pore size transwell chambers (Corning, USA). Cells were plated in the upper chamber and completed medium was added as a chemoattractant in the lower chamber. The upper chamber was coated with 30 μg of Matrigel (BD Biosciences, USA). After 24 hours of incubation, cells entering the lower chamber were fixed in the 4% paraformaldehyde and stained with crystal violet. We randomly selected five microscopic fields to count the cell number and captured the images under the microscope.

To further examined the cell invasion and migration, the transwell invasion and migration was performed using 8 μm pore size transwell chambers (Corning, USA) in vitro following the manufacturer's instructions. In brief, the matrigel (5 mg/ml, Corning, USA) was diluted into 1 mg/ml in ice-cold RPMI 1640 medium supplemented with 10% FBS. An aliquot of 200 μL diluted matrigel was added to the upper transwell chambers and incubated at 37°C for 4 h for gelling. A total of 1 × 10⁵ cells in 400 μL media supplemented with no FBS were plated in the upper chamber and 600 μL RPMI 1640 medium supplemented with 10% FBS was covered on the bottom chambers as chemo attractant. After incubation at 37°C for 48 h, the non-invasive cells in the top surface were carefully removed with a cotton swab. The invasive cells that had traversed to the bottom surface were fixed in dehydrated alcohol for 30 min and stained with 4 mg/ml crystal violet for 10 min. To quantify the traversed cells, cell counting was obtained by photographing 5 random fields under microscope at 400× magnification [27 (link)]. The migration assay was performed in a similar strategy with chamber membrane without coating with matrigel.