Polyphosphate formaldehyde

The invasion activity of tumor cells was evaluated by their ability to pass through a gel matrix (Matrigel; BD, Franklin Lakes, NJ, USA). Briefly, Matrigel solution was diluted with FBS-free medium at a proportion of 1:5 to coat the 6.5 mm diameter polycarbonate filters (8 μm pore) of the Transwell chambers (Corning, NY, USA) in 24-well plates. With 40 ml working solution in every filter, the plates were placed at 37 °C for more than 5 h to solidify. Tumor cells were seeded at a density of 2 × 10⁵ per chamber and cultured with FBS-free medium in the upper compartments of the chamber for 48 h while the lower compartment was filled with complete medium. Non-invasive cells on the upper surface of the filter were wiped off using a cotton swab, while the cells adhering to the lower surface were fixed using 4% polyphosphate formaldehyde (Beyotime, Shanghai, China). These cells with stronger invasion potential were counted after crystal violet staining (Beyotime). The experiments were repeated for three times independently.

Invasion activity of cells in vitro was examined by their ability to pass through a gel matrix (Matrigel; BD; Franklin Lakes, NJ, USA). In brief, 6.5 mm diameter polycarbonate filters (8 μm pore) of the transwell chambers (Corning; NY, USA) for use in 24-well plates were coated with 65 ml of working Matrigel solution (1:5 diluted with FBS-free DMEM) and were placed at 37°C for at least 0.5 h to dry. Cells were subjected to different treatments as indicated and were then seeded at a density of 2 × 10⁵ per chamber, and FBS-free DMEM was added into the upper compartments of the chambers. Meanwhile, the lower compartments of the chamber were filled with DMEM containing 10% FBS. After 48 h, non-invasive cells on the upper surface of the filters were removed completely by wiping the filter surface with cotton swabs. Viable invasive cells adhering to the lower surface of the filter were fixed using 4% polyphosphate formaldehyde (Beyotime; Shanghai, China). The invaded cells were stained using crystal violet (Beyotime; Shanghai, China). The number of stained cells in five 100× vision fields of each treatment was counted. Average cell numbers of these five fields were calculated and then normalized to control. All experiments were carried out in triplicate and independently repeated three times.