Transwell cell culture chambers with 8 μm pore filters

Migration assays were performed using Transwell cell culture chambers with 8-μm pore filters (Corning Life Sciences). Following treatment with various concentrations of PZH under normoxia or hypoxia for 6 h, HCT-8 cells were trypsinized and resuspended in serum-free RPMI-1640. A total of 5×10⁴ cells in 200 μl serum-free RPMI-1640 were plated in the upper chambers. RPMI-1640 media containing 10% (v/v) FBS was used in the lower chambers as a chemoattractant. Cells were allowed to migrate for 12 h under normoxia, following which the non-migrating cells were removed from the upper surface of the Transwell membrane in each Transwell using a cotton swab. Membranes were then stained with crystal violet. For quantification, the average number of migrating cells per field was assessed by counting three random fields under a Leica phase-contrast microscope (Leica, Microsystems Ltd.) at a magnification of ×200. For the cell invasion assays, the procedure was the same as that used for the migration assay; however, the upper chambers were coated with 100 μl/well 0.2 mg/ml Matrigel Matrix (BD Biosciences) and cell invasion was allowed to progress for 24 h in normoxia.

Cell migration assays were performed with transwell cell culture chambers with 8 μm pore filters (Corning, NY, USA). After treatment with various concentrations of PZH under normoxia for 24 hours, HUVECs cells were trypsinized and resuspended in serum-free RPMI 1640. HUVECs (5 × 10⁴ cells in 200 μL of serum-free RPMI 1640) were added to the upper compartment in duplicate filters and RPMI-1640 media containing 10% (v/v) FBS were used as a chemoattractant in the lower chambers. Cells were allowed to migrate for 12 hours in normoxic/hypoxic conditions. The nonmigrating cells were removed from the upper surface of each transwell by cotton swab. Transwell membranes were then stained with crystal violet. For quantification, the average number of migrating cells per field was assessed by counting 5 random fields under a phase-contrast microscope (Leica, German) at 200x magnification.

Cell migration was further assessed via Transwell cell culture chambers with 8 μm pore filters (Corning Life Sciences, Corning, NY, USA). Following an incubation with QFG (0, 0.5, 1, and 2 mg/mL) for 24 h, surviving HCT-8 and HCT116 cells were further seeded into the upper chambers at a density of 5 × 10⁴ cells/chamber. Culture medium containing 10% FBS was added to the lower chambers as a chemoattractant. Some cells migrated towards the complete medium within 12 h and were stained with crystal violet for 15 min at room temperature. Cells in the upper chamber that had not migrated were removed using wet cotton swabs. To calculate the average number of migrated (stained) cells per field, three fields were randomly selected using a phase contrast microscope (Leica Microsystems GmbH, Germany) at a magnification of 200× to count the stained cells. The setup for cell invasion assays was the same as that for cell migration, except that the upper chambers contained Matrigel matrix (BD Biosciences, Franklin Lakes NJ, USA).