C8470

A transwell assay was performed in a Modified Boyden Chamber (Transwell, Costar, 3422, Cambridge, MA) to evaluate cell migration and invasion. Pancreatic cancer cells (SW1990 and ASPC1) in the logarithmic growth phase were trypsinized and inoculated into a six-well plate at a density of 300,000 cells/well. The cells were cultured for 24 h, followed by a transwell assay after 48 h of transfection. Prior to cell inoculation, the 24-well plates and transwell chambers were soaked for 5 min with phosphate-buffered saline. For the invasion assay, the chambers were additionally coated with 80 µl of Matrigel for 30 min in an incubator at 37 °C. Finally, the cells were starved for 24 h in serum-free medium, and 0.3 ml of cell suspension (200,000 cells/ml) was seeded in each upper transwell chamber; each lower chamber was filled with 0.7 ml of 10% FBS-DMEM. After 72 h of incubation at room temperature, cells that had migrated and invaded the lower chamber were fixed for 10 min with 1 ml of 4% formaldehyde (SF877501, Shanghai Sinopharm, China) and stained with 0.5% crystal violet (C8470, Solarbio) for 30 min. After removing the non-migrating and non-invading cells, the migrated and invaded cells were counted in at least three random fields under a microscope at 200 × magnification.

The cells were digested with trypsin, and then, resuspended in serum‐free medium (1 × 10⁵ cells/ml). Next, 150 μl of cell suspension of each group was added to the upper chamber of the Transwell chamber (3428, Corning, USA) pre‐coated with Matrigel (354230, BD, USA), and 500 μl of complete medium containing 10% of FBS was added to the lower chamber. After 48 hours of incubation, the chamber was removed, and then, the cells on the membrane were fixed with 950 ml/L of ethanol and stained with 0.1% of crystal violet solution (C8470, Solarbio, China). Finally, the invasive cells from five randomly chosen fields of each sample were counted under an inverted microscope (magnification ×200).

CV assay is the most commonly used quantitative technique for detecting biomass accumulation in microplate method. Specimens (n=5 per group) with C. albicans biofilms were rinsed with PBS to remove nonadherent fungi and then air dried for 20 min. The air-dried specimens were submerged in 100% methyl alcohol for 15 min for fixation and then stained with 0.1% CV solution (C8470, Solarbio, China) for 15 mins. The bound dye was extracted from the stained cells with 95% ethanol solution. Biomass accumulation was then quantified by measuring the optical density of the CV/ethanol extract at a wavelength of 600 nm in a microplate reader (SpectraMax M5, Molecular Devices, USA).