Transwell chamber with 8 μm pore size

Transwell chamber with 8 μm pore size (Corning Incorporated, USA) was adopted to estimate the cell migration of ARPE-19 cells. Briefly, 500 μl DMEM containing 2 × 10⁵ treated ARPE-19 cells were added into the upper chamber, whereas 500 μl serum including (10%) DMEM was added into the lower one. After 24 h of culture, cells remained in the upper surface were discarded and those cells on the undersurface were fixed and stained with 5% crystal violet followed by number counting manually.

Migration of early OPCs was performed using a 12-well Transwell chamber with 8 μm pore-size (Corning, NY, USA). Generated early OPCs were plated on the upper wells at 40 × 10⁴ cells/mL with OPC fasting medium (NeuroBasal medium with 100 units/mL penicillin and 100 μg/mL streptomycin; all from Thermo Scientific, Waltham, MA, USA), and 500 μL of 24 h CM recovered from either CT or SPMS-derived early OPC cells and of indicated factor: NG2 3 ng/μL (R&D Systems, Minneapolis, MN, USA), laminin 10 ug/mL (Thermo Scientific, Waltham, MA, USA), or bFGF 5 ng/mL (Thermo Scientific, Waltham, MA, USA), was added to lower well of the chamber. After 24 h of culture at 37 °C, cells on the upper surface of the membrane were removed with a cotton swab, whereas migrated cells on the lower membrane surface were fixed in 4% paraformaldehyd for 15 min and stained in 0.5% crystal violet aqueous solution (Sigma–Aldrich, St. Louis, MO, USA) in 20% methanol for 20 min, rinsed 3× with H2Odd, immersed in methanol for 15 min to solubilize the dye. The absorbance of the extracted solution was read (OD 560).

Transwell^® chamber with 8 μm pore size (Corning, Corning, NY) was used for the cellular migration/invasion assay. After transfection with various plasmids for 48 hrs, cells were harvested and resuspended in serum-free medium, then the cells (1.5 x 10⁴) were added to the upper chamber with uncoated polycarbonate membrane for migration assay, or with matrigel-coated (BD Bioscience, Bedford, MA) membrane for invasion assay, respectively. RPMI1640 medium supplemented with 10% FBS were placed into each well of the bottom chamber to act as a chemoattractant. After incubation for 12 hrs at 37°C, cells on the upper side of membrane were removed by a cotton swab. The migrating cells to the bottom surface of the membrane were fixed with 100% methanol for 10 mins, stained with 10% Giemsa for 30 mins, and counted under a microscope in 5 random fields (100 X) per well and then quantified by a software Image-J. Values are means±SD for three determinations.