4.5. Cell Migration Assay

A wound-healing assay examined the effect of HXL131 on PC3 cell migration, and this was performed by us referring to the method that is described in [59]. Briefly, the cells were seeded at 1 × 10⁶ to 6-well plates using an FBS-free medium. When the confluence was greater than 90%, a sterile 200 µL pipette tip was used to create a wound in the PC3 cell monolayer, and detached cells were gently washed with PBS. Next, different concentrations of HXL131 were added. To avoid cell migration inhibition that is caused by high concentrations of HXL131, we used the HXL131 with a lower concentration gradient (1.25, 2.5, and 5 µmol/L). Images were taken at 0, 24, and 48 h after the scratch, and the wound-healing rates in each group were calculated using Image J software (V1.8.0.112, NIH, Bethesda, MD, USA).

The trans-well chamber detected the effect of HXL131 on PC3 cell migration. The cell density was adjusted to 2 × 10⁵ cells/mL in the DMEM without FBS. A two hundred µL cell suspension was added into the upper chamber of the trans-well chamber, and 600 μL DMEM containing 10% FBS was added into the lower chamber. After the cells had adhered to the wall, HXL131 (1.25, 2.5, 5 µmol/L) was added into the upper chamber and incubated for 24 h. The cells that did not migrate to the bottom of the trans-well chamber were wiped with cotton swabs, soaked in 70% methanol for 20 min to fix the migrated cells, stained with 0.1% crystal violet for 20 min, and washed with PBS and dried. The number of migrated cells was randomly observed under the microscope and photographed in three fields. The formula for the migration of the cells is as follows: inhibition rate of cell migration = 1 − (Average number of migrated cells in the experimental group/Average number of migrated cells in the control group) × 100%.

The effects of HXL131 on PC3 cell migration were monitored using RTCA technology. Referring to the protocol in [60], PC3 cells were cultured in advance with FBS-free DMEM to induce the cell starvation effect. One hundred and sixty-five µL DMEM containing FBS was added to the lower chamber of the RTCA CIM-plate 16-well Plate to induce chemotaxis, and 30 µL DMEM free FBS was added to the upper chamber, and this placed in an incubator for 1 h to soak the membrane of the chamber. The cells were collected and inoculated into the upper chamber of the detection plate with 6 × 10⁴ cells/well and incubated at room temperature for 30 min. HXL131 (2.5, 5, 10, 15 µmol/L) was added as the experimental group, and 0.1% DMSO was used as the control group. The cell migration process of PC3 was monitored using the xCELLigence RTCA DP cell analyzer, and the real-time kinetic curve of cell migration was obtained.