Boyden chamber system

A Boyden chamber system (Costar, Corning, Inc., Tewkesbury, MA, US) was used for the transwell migration assay. The SGC-7901 and HGC-27 cells (5×10⁴) were transfected with either 25 nM miR-141 or negative control in 24-well plates for 24 h. The cells were then trypsinized and 10⁴ cells were seeded into each insert in culture medium, and the same medium was placed in the well below. Following a 24 h incubation period, the cells remaining in the top inserts were removed using a cotton swab, and the cells that had migrated through the filter were fixed with 75% ethanol for 30 min, followed by 0.1% crystal violet staining (Sigma-Aldrich, St. Louis, MO, USA) for 20 min. The ability of the cells to migrate to the lower chamber was visualized, and the images were captured using an inverted microscope (Leica DMI 4000B, Leica Microsystems, Wetzlar, Germany).

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.

As previously reported, primary OPCs were seeded in an 8 µm pore-sized transwell-based Boyden chamber system (Corning Incorporated, NY, USA) to perform the OPC migration assay (32 (link)). Scramble or nexilin siRNA (2 ng/ml, Santa Cruz) was added and cultured with the OPCs for 1 day prior to analysis. OPCs were seeded in the upper chamber (pre-coated with poly-d-lysine on both sides) at 100,000 per well and maintained in 200 µl of neurobasal medium supplemented with Ara-C (5 µM, Sangon Biotech, China) to prevent proliferation. PDGF (10 ng/ml, Sangon Biotech) was added to the lower chamber to induce the migration of OPCs for 24 h. The migrated cells on the lower chamber were counted in six randomly selected microscopic fields from each well. Then, these cells were fixed with 4% PFA and subjected to immunofluorescence assessment.