24 well transwell chambers with 8 μm pores

The 24-well transwell chambers with 8 μm pores (Millipore, Billerica, USA) were used for transwell assays. HeLa cells were seeded into the upper chambers at a density of 1 × 10⁵ and incubated with FBS-free DMEM dissolved with various concentrations of flavonoids in A. conyzoides (50, 200, and 400 μg/mL) or sterile ddH₂O (control). The lower chambers contained the DMEM supplemented with 10% FBS. After incubation for 48 h, cells on the internal surface of the upper chambers were washed with PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet (Beyotime, Shanghai, China) for 20 min, and then rinsed with PBS for three times. Finally, three random views for each chamber were captured using an inverted optical microscope-camera system (Olympus), and the number of migration cells in each view was counted. Three independent experiments were performed.

Transwell assay was performed with the 24-well transwell chambers with 8 μm pores (Millipore, Billerica, USA) as previously described, with some modifications [27 (link)]. Briefly, before seeding cells, the upper chamber was added with 100 μL matrigel (BD, USA) diluted in RPMI-1640 medium at the ratio of 1:8 and maintained at 37°C for 6 h. Then, the transfected A549/DDP cells were plated into the upper chamber at a density of 1 × 10⁵ and cultured in FBS-free RPMI-1640 medium dissolved with cisplatin (20 μM). RPMI-1640 medium containing 10% FBS was added into the lower chamber. After being cultured at 37°C for 48 h, the invaded cells on the lower surface of the membrane were fixed with 4% paraformaldehyde (Boster, Wuhan, China) for 30 min, stained with 0.1% crystal violet (Beyotime) for 15 min and then washed thrice with PBS. Finally, the invaded cells were viewed and photographed using an inverted optical microscope (Olympus). The experiments were performed in triplicate.