4.10. Cell Migration Assay

MS-5 stromal cells were seeded 24 h in advance as described above, and conditioned medium from the culture (CM) was collected at the time of experiment. JeKo-1 and REC-1 cells (cell density of 0.8 × 10⁶ cells/mL) were stained with calcein-AM (1 µM; Thermo Fisher Scientific, NY, USA) for 30 min at 37 °C, 5% CO2. Cells were washed twice and resuspended to 2 × 10⁶ cells/mL density in either 10% FBS in αMEM-glutamax in the case of chemotaxis towards CM or in 10% FBS in RPMI-glutamax in the case of chemotaxis towards CXCL12. Chemotaxis assay was performed using Boyden chamber system (Corning, NY, USA), during which cells were placed on top of the Fluoroblok insert (8 µm pore size; Corning, NY, USA) with the well below containing either medium alone, CM or medium with CXCL12 (200 ng/mL; R&D Systems, Minneapolis, MN, USA). Cumulative numbers of migrated cells were acquired using a Nikon Eclipse Ti confocal microscopy system (Nikon Instruments, Melville, NY, USA) with a 10× objective. Migration was quantified by counting fluorescent cells in bottom wells with NIS-Elements AR software (Nikon Instruments, Melville, NY, USA) either in a time lapse manner or at a single time point (4 h). When indicated, cells were treated with AMD3100 (25 µg/mL) (Sigma, St. Louise, MO, USA) 20 min prior to placing the cells in the upper chamber.