Crystal violent

As outlined above, H1299 cells were transfected with siRNAs and/or miRNAs, and 1000 cells were seeded per well in 6-well plates. After 48 h, cells were switched to a complete RPMI-1640 medium. Then the cells were incubated in a CO₂ incubator at 37 °C for another one week. After a total of 9 days, the cells were fixed with methanol at room temperature for 30 min and stained with 0.5% crystal violent (Sigma, St. Louis, MO, USA) with 25% methanol at room temperature for 1 h. The plates were then washed under running water and dried. The number of colonies per well were counted. A colony was defined as a group of at least 50 cells under the microscope.

A chemoinvasion assay was performed to evaluate the ability of cells to cross a Matrigel membrane. The upper chambers, with 8 mm pores, were coated with 50 μl Matrigel diluted 1:10 (v:v) in DMEM and were incubated at 37°C for 4 h. The lower chambers contained either DMEM supplemented with 1% BSA as a control or UC-CM. For specific factor blocking assays, 20 μg/ml each of the monoclonal mouse anti-human anti-SDF-1 (cat. no. MAB350; R&D Systems, Inc.), anti-MCP-1 (cat. no. 16-7096; eBioscience, Inc.) and anti-HGF (eBioscience, Inc.) antibodies were added to the lower chambers. The fibroblasts, HUVECs and UC-MSCs were prepared in DMEM supplemented with 1% BSA, and 5×10⁴ cells in 0.5 ml suspension were added to each upper chamber. Each experiment was performed in triplicate. The chambers were placed in a 24-well plate and were incubated at 37°C, with 5% CO₂ for 24 h. The cells, which had not crossed the membrane were removed with a wet cotton bud. The undersides of the filters were then fixed in methanol (Sigma-Aldrich) for 10 min and stained with 0.1% crystal violent (Sigma-Aldrich), and images of the cells, which had invaded to the underside of the insert were captured. Three random fields were selected (magnification, ×40) by microscopy (CKX31; Olympus Corporation) and counted.

The 24-well transwell apparatus with 8 μm pores (Costar, Dallas, TX, USA) was utilized for the migration assay. After transfection or infection, the cells were resuspended in serum-low medium (0.5% FBS) at a density of 1 × 10⁶ cells/ml and seeded into the upper chambers with 200 μl of starved medium. The lower chambers were filled with 500 μl of serum-free medium in the presence or absence of PDGF-BB (30 ng/ml). After cultivation for 24 h, the cells that migrated through the membranes were fixed with 4% formaldehyde (Sigma) and stained with crystal violent (Sigma). Images were taken under a microscope (Olympus), and the number of migrated cells was counted.