We investigated cell migration using wound healing and Transwell assays. For the wound healing assay, CFs were seeded in six-well plates. Wounds were made through the cell monolayer using 1000-mL plastic tips after the cells were incubated for 12 h, and images were captured using a Nikon Ti-S inverted phase-contrast microscope (Nikon, Tokyo, Japan) at 24 h to calculate the healing rates.
For the Transwell assay, the cells (1 × 105 cells) were trypsinized and seeded into the upper chambers (8-mm pores, 24-well, Corning Life Sciences, Corning, NY, USA) in FBS-free DMEM (200 mL). The lower chambers contained DMEM supplemented with HG or the OC, and incubation was performed for 24 h. Next, the cells that migrated into the lower chambers were fixed using 4% paraformaldehyde after removal of the medium and stained with crystal violet for 0.5 h. Images of five stochastic fields per membrane were obtained using a Nikon Ti-S inverted phase-contrast microscope.
For the Transwell assay, the cells (1 × 105 cells) were trypsinized and seeded into the upper chambers (8-mm pores, 24-well, Corning Life Sciences, Corning, NY, USA) in FBS-free DMEM (200 mL). The lower chambers contained DMEM supplemented with HG or the OC, and incubation was performed for 24 h. Next, the cells that migrated into the lower chambers were fixed using 4% paraformaldehyde after removal of the medium and stained with crystal violet for 0.5 h. Images of five stochastic fields per membrane were obtained using a Nikon Ti-S inverted phase-contrast microscope.