24 well transwell plate

Cell migration and invasion assay was performed using 24-well transwell plates (8.0 μm pore size; Millipore, USA) precoated without or with matrigel (BD Biosciences, USA). SMMC7721 cells (5 × 10⁴) were suspended in 1.5 ml serum-free DMEM media and transferred into the inside chamber of a 24-well cell culture insert with 8.0 μm pore size. 600 μl media with 20% FBS was added into the outside well. After 24 h incubation, cells remaining on the upper side of the filters were cleaned with cotton-tipped swabs. Cells on the lower surface of the membrane were fixed by methanol and subjected to Giemsa staining. The cells on the underside of the filters were counted at five randomly selected fields (at 200 × magnification), and the average cell number per view was calculated. All experiments were performed in triplicate.

Transwell migration assays were performed, as previously described (12 (link)). Briefly, the cells were seeded into a 24-well Transwell plates (Millipore, Bedford, MA, USA) in 10% FBS medium at a density of 1×10⁵ cells/well. Following incubation for 24 h at 37°C, the medium was replaced with serum-free medium and the cells were treated with 20, 40 or 80 µM of luteolin or 300 nM Taxol for 24 h at 37°C, while RPMI-1640 medium, containing 10% FBS was added to the lower chamber. The NCI-H460 cells were treated with the drug and cultured at 37°C for a further 5 h. The non-adherent cells were removed by washing with PBS, and the adherent cells were fixed in ethanol. Following staining with 0.1% crystal violet (Tokyo Chemical Industry, Tokyo, Japan), images were captured by microscopy (IX81; Olympus, Tokyo, Japan).

We performed cell migration and invasion assays with 24‐well transwell plates (8.0‐μm pore size, Millipore, Billerica, MA, USA) with or without Matrigel coating (Corning, NY, USA). Cells were cultured with serum‐free DMEM in the upper chamber, and DMEM supplemented with 10% FBS was added to the lower chamber. After 12 and 24 hours of incubation, we removed the residual cells in the upper chamber with cotton swabs and fixed the filters using 4% paraformaldehyde for 15 minutes prior to staining with 0.1% crystal violet. Cells in ten random optical fields from triplicate filters were counted and averaged.

To assess migration, quiesced VSMCs were treated with or without VOSO₄ or NaVO₃ (1 µg/mL) with or without inhibitor for 24 h. VSMCs were trypsinized, washed with PBS, re-suspended in medium with 0.5 % FBS, and then placed in the upper chamber of 24-well transwell plates (Millipore, 8-μm pore size) in triplicate (20,000 cells/well). The bottom chambers were filled with starvation medium containing PDGF-BB (Peprotech, 10 ng/mL) as a chemoattractant. After 4 h, the upper layer was scraped by cotton swab to remove non migratory cells, membrane fixed, and stained with crystal violet solution (0.1% crystal violet, 20% ethanol, and 1% formaldehyde in ddH₂O). Cells that had migrated to the underside of the membrane were visualized by microscope. The crystal violet in the cells was extracted by crystal violet extraction buffer (50% ethanol and 0.1% acetic acid in ddH₂O) and read by a microplate reader at absorbance 595 nm. The data were plotted as the fold change versus vehicle, arbitrarily set to 1.

For the scratch wound-healing assay, cells at logarithmic growth phase were transfected with siRNA or treated with pcDNA3.1-Meox1. Then, 2 × 10⁵ cells were seeded into six-well plates and cultured to 70% confluence. Scratch wounds were created in the monolayer (0 h) with 1 mL pipette tips and then captured every 12 h using a Zeiss video microscope Carl Zeiss AG is in Jena, Germany. The wound-healing distance was measured with ImageJ v1.48 software (National Institutes of Health, Bethesda, Maryland, United States).
Transwell assays were carried out with 24-well Transwell plates (8-μm pore size; Millipore, Bedford, Massachusetts, United States); 1 × 10⁵ cells transfected before were seeded on the upper chamber in serum-free medium, whereas the lower chamber contained medium with 20% FBS applied as a chemoattractant. After incubation for 24 h, the cells on the bottom surface of the filter were fixed with 4% paraformaldehyde, stained with hematoxylin-eosin dye, and counted.