8 μm transwell chamber

Twelve-well 8 μm Transwell chambers (BD Biosciences) were employed to evaluate migration and invasion of AGS or HGC27 cells. The transfected STAD cells were counted and prepared into cell suspensions at a density of 2 × 10⁵ cells/mL. For migration assay, 200 µL of cell solution was dispensed into the top compartment, while 800 µL of chemical induction agent (RPMI1640 medium + 20% FBS) was added to the basolateral compartment. After being maintained in the incubator for 24 h, the top compartment was removed and immersed in methanol for 20 min to fix the cells, followed by 0.1% crystal violet staining for 30 min (room temperature). Residual cells on the upper surface of the upper chamber were manually wiped off with a wet cotton swab. At least 4 random fields of view were selected for photographing and counting under a 200×Olympus inverted microscope. For the invasion assay, 50 µL of Matrigel solution needs to be pre-coated in the upper chamber of the Transwell and incubated to solidify. Other steps are similar to the migration assay. For the invasion assay, 50 µL of matrix gel solution needed to be spread on the Transwell upper chamber and incubated to solidify, and other operational steps were similar to migration experiments.

A total of 5 × 10⁴ cells/well in serum-free DMEM were seeded into the upper chamber of an 8-μm transwell chamber (Beckton Dickinson, Franklin Lakes, NJ, USA); DMEM with 10% bovine serum albumin (BSA) was added in the lower chamber. After 24-h incubation at 37 °C, the cells in the upper chamber were fixed in methanol and then stained with Giemsa solution (Beyotime, Nantong, China). Then, the migrated cells were imaged and quantified.

This assay was performed as described previously [19 (link)]. Migration and invasion experiments in vitro were performed in the 24-well plate of 8-μm transwell chamber (BD Biosciences, USA) with or without Matrigel (1:3 mixed with PBS; BD Falcon^™). Cells transfected (1 × 10⁵ cells per well) were suspended in 100μl serum-free medium and then plated onto the top chamber in the 24-well plate, and the lower chamber of each well insert was filled with 600μl serum-containing medium. After 24h of incubation at 37°C, the cells that migrated or invaded into the lower chambers were fixed with 4% paraformaldehyde, washed with PBS, stained with crystal violet and then counted under a light microscope (Olympus, Tokyo, Japan) at ×100 magnification in five randomly selected fields across the center and the periphery of the membrane. Experiments were performed in triplicate.

Transwell assay was carried out by using 8 μm transwell chamber according to the manual (BD Science). 1×10⁵ cells per well were digested and washed with serum-free DMEM for three times before seeding into the upper chamber. Serum-free DMEM was used to dissolve fucoidan to 0.5 mg/mL and the cells were resuspended at a volume of 100 μL per well and planted into the upper chamber, while 600 μL DMEM supplemented with 20% FBS and 0.5 mg/mL fucoidan was added into the lower chamber. After 48 h, the films were removed, washed with PBS twice and fixed with 4% paraformaldehyde for 15 min. 0.1% crystal violet was used to stain the film for 15 min and then the upper side of the film was carefully wiped with cotton swabs. The cells were recorded with microscope and the total area of cells was counted by ImageJ and values were presented as the mean ± standard deviation (SD). Each group had three independent repetitions.