Upper transwell chamber

Transfected A704 cells were harvested, suspended with serum-free MEM medium, and seeded at a density of 4 × 10⁴ cells per well into the Transwell upper chambers (8 μm; Millipore, Billerica, MA, United States) coated with Matrigel (BD, Bedford, MA, United States). Then, 700 μl complete media was added to the lower chambers. After incubation for 48 h at 37°C in a 5% CO₂ incubator, cells were fixed by methanol and stained with 0.1% crystal violet. The invasive cells were counted in five random fields (×100) for quantification after wiping off non-invasive cells of the upper chambers. The assays were performed in triplicate.

Pretreated cells (1 × 10⁴ in 200 µl medium without FBS) were placed in the Transwell upper chamber (6 µm pore size; Millipore) chambers in 24-well plates while 600 µl of medium containing 20% FBS were placed in the lower chamber, and incubated at 37 °C. At specified times, cells on the lower part of the membrane were removed, fixed (4% paraformaldehyde), and stained (0.1% crystal violet). The remaining membrane was washed in PBS, air-dried, and the adherent cells counted under light microscopy (×400 magnification), using the average of cell numbers in five randomly selected fields.

Firstly, HUVECs (1 × 10⁶) were seeded into transwell upper chamber with pore size of 0.4 μm (Millipore, Billerica, MA, USA) without matrigel. Equal amounts of SW480 and HCT116 cells of each group following transfection were inoculated into the 24-well plates in the lower chamber. After 24 h co-culture (5% CO₂, 37 ℃), HUVECs were used for the subsequent assays.
HUVEC cells were inoculated into 6-well plates with a density of 2 × 10⁶ /well and maintained in incubator (5% CO₂, 37 ℃) for 24 h. Subsequently, cells grown in monolayer were scratched with a sterile 200 μl pipette tip, followed by PBS washing and subsequent incubation in 5% CO₂ at 37 ℃ for 24 h. The scratch distance after culture for 0 h and 24 h was observed and photographed under a microscope. HUVEC cell migration rate was calculated as follows: Migration rate = (0 h scratch distance—24 h scratch distance)/0 h scratch distance.

The chemotaxis of BMDMs was assessed in vitro using a transwell chamber migration assay. BMDMs (1×10⁵ cells) were plated onto a transwell upper chamber (8-μm pore size, Millipore) and allowed to migrate across the porous filter for 4 h at 37° C towards CCL2 (50 ng/mL, PeproTech, Inc.). Migrated BMDMs on the bottom of the filter were stained with crystal violet and counted using a fluorescence microscope. The number of cells was determined in five random fields (magnification × 200) for each experiment.

Wound healing and Transwell experiments were employed to determine the migration ability of MDA-MB-231 cells. For wound healing assay, a linear wound was sketched with the pipette tip after cells reached a confluency of 90%-95%. Then cells were incubated in serum-free medium for 24 h and 48 h. Cells were then photographed at different time points and the migration rate was calculated by measuring the wound area. The wound areas at 0 h and 48 h were calculated by Image Pro Plus software. The healing rate was calculated using the formula: [wound area (0 h) - wound area (48 h)]/wound area (0 h). The transwell assay was also used to assess migration ability. Cells were plated into Transwell upper chamber at a density of 1x10⁵ cells/chamber (Millipore, Germany), and cultured with fresh medium without FBS. The lower chamber was surrounded with 600 μl of 10% FBS. After incubation for 12 h, cells on the upper chamber were wiped off and fixed for 30 minutes with ethanol. Then cells were stained with 0.1% crystal violet for 20 minutes after washing with PBS buffer for twice. Then, the migrated cells was calculated using a microscope (OLYMP^®S, IX51, Japan).