Transwell cell culture inserts with 8 μm pores

Six-well Transwell cell culture inserts with 8-μm pores (Corning) were used to isolate muscle invasive and nonmuscle invasive 5637 and T24 cell sublines. Generally, 5 × 10⁵ cells were firstly cultured in serum-free RPMI-1640 medium for 24 h and then suspended and seeded into the top chamber coated with 200 mg/mL of Matrigel (BD Biosciences). To stimulate cell penetration with a chemotactic gradient, the lower well was filled with 2.5 mL RPMI-1640 medium containing 20% bovine serum. 24 h after incubation, the noninvasive cells on the topside and the invasive cells on the bottom side of the polycarbonate membrane were harvested, respectively, for further cultivation. After 10 rounds of selection, the stable invasive cell sublines were designated as 5637-M and T24-M and the stable noninvasive sublines were designated as 5637-NM and T24-NM, respectively.

24-well Transwell cell culture inserts with 8 μm pores (Corning) was used to assess the invasive ability of the cell sublines. 5 × 10⁴ cells firstly cultured in serum-free RPMI-1640 medium for 24 h was suspended and seeded into the top chamber coated with 200 mg/mL of Matrigel (BD Biosciences). To stimulate cell penetration with a chemotactic gradient, the lower well was filled with 2.5 mL RPMI-1640 medium containing 20% bovine serum. After 24 h incubation at 37°C, the topside of the membrane was wiped with cotton wool to remove noninvasive cells. The invading cells on the bottom side were fixed by using 100% methanol for 10 min and then dried under room temperate and stained with 0.1% crystal violet for 20 min. The number of cells was counted under a microscope with 100x magnification. Each test was performed in triplicate.

Cell invasion assay was performed using Transwell cell culture inserts with 8 μm pores (Corning, NY, USA) in accordance with the manufacture’s introduction. The matrigel (Corning, NY, USA) was added to the inserts 2 h before cells were plated into inserts. In brief, 1 × 10⁵ cells suspended in 100 μl of serum-free medium were seeded onto the upper chamber, and 500 μl of the same medium containing 10% FBS was placed in the lower chamber. After incubation for 24 h, cells were washed with PBS, fixed with 4% formaldehyde, and stained with crystal violet for 25 min. Cells remaining on the upper surface of filter membrane were completely removed by wiping with a cotton swab before fixed. Invaded cells were then counted from six random fields under a light microscope.

Cell migration assay was performed using Transwell cell culture inserts with 8 μm pores (Corning). The matrigel was added to the inserts for 4 h before cells were plated into inserts. Dissociated cells (5× 10⁴/insert) in serum free medium were seeded on inserts and medium 10 % FBS was added to the lower chambers. After incubation for 36 h, and the non-migrating cells on the upper membrane of insert were erased by cotton swab. The migration cells adhered to the membrane lower surface were fixed with cold 100 % formaldehyde for 20 min, stained with hematoxylin for 25 min and then number of cells was counted under a microscope in five random optical fields.