Upper transwell chamber

In order to measure the migration of the EPCs, 1.5×10⁵ cells at passage 4, 6 and 8 with or without pre-treatment with tyrphostin AG1295 (Sigma-Aldrich) at 20 µM for 1 h were seeded in the upper Transwell chamber (BD Biosciences) in serum-free medium, with 500 µl DMEM with 10% FBS in the lower chamber. After 24 h, cells that did not migrate through the pores were carefully wiped out with a cotton-tipped swab. The filters were fixed in 90% alcohol, followed by staining with 0.1% crystal violet (Meryer, Shanghai, China). After washing with PBS 3 times, the filters were observed under an inverted microscope (Olympus).

In preparation for the motility assay, SiHa cells were resuspended at a density of 1×10⁵ cells/ml in serum-free DMEM. The cell suspension (200 µl) was added to different concentrations of β-elemene (0, 20, 30 and 40 µg/ml) and placed in an upper Transwell chamber (BD Biosciences). Simultaneously, 600 µl conditioned medium containing 20% FBS, in addition to the different aforementioned concentrations of β-elemene, was added to the bottom Transwell chamber. Following a further 24 h incubation period at 37°C, the SiHa cells that had migrated to the bottom chamber were fixed in methanol for 30 min at room temperature, stained with crystal violet for 30 min at room temperature, and counted using a light microscope under a 10-fold mirror vision. In order to perform the invasion assay, 40 µl Matrigel (0.5 mg/ml; Beckman Coulter, Inc., Brea, CA, USA) was spread onto the upper Transwell chamber and incubated for 4 h at 37°C. The plating of the lower chamber was performed in accordance with the aforementioned protocol. Following this, the cells were incubated for 72 h at 37°C in the Transwell device and then fixed, stained and counted according to the aforementioned protocol. Each assay was performed in triplicate.

Cells at a density of 1×10⁵ cells/ml were seeded in the upper Transwell chamber (BD Biosciences) precoated with Matrigel (BD Biosciences). After 48 h of incubation, the cells on the upper surface of the microporous membrane were wiped off with a cotton swab, and the remaining cells were stained with 0.5% crystal violet (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min at room temperature and photographed at a magnification of ×200 using an Olympus IX71 inverted microscope (Olympus Corp., Tokyo, Japan). The absorbance at 570 nm was measured using a Tecan Infinite F500 microplate reader (Tecan Group AG, Männedorf, Switzerland).

In the migration assay, cells were treated for 48 h with nanoparticles, then 2 × 10⁴ cells were resuspended in 300 μL cell culture medium with 0.5% FBS and placed in the upper transwell chamber (8 μm pore size, BD Biosciences). The upper chamber was placed in a 24-well culture dish containing 1 mL of complete cell culture medium (with 10% FBS). After 48 h incubation, non-migrated cells on the upper membrane were removed with a cotton swab. Migrated cells on the bottom surface were fixed with methanol (−20°C) and stained with 0.5% crystal violet. Four fields of each well were photographed, and the cells were counted. In the invasion assay, Matrigel-coated transwell chambers (BD Biosciences) were used. Percentage invasion was calculated as the number of invaded cells in comparison with the number of migrated cells. All experiments were repeated three times.