8.0 μm pores

To observe the ability of hNSCs to migrate to melanoma and normal human lung cells, both A375SM and L132 (1×10⁵ cells/well) were plated in a 24-well plate containing 5% CD-FBS phenol free DMEM and incubated for 24 hours. The bottom surface of the transwell with 8.0-μm pores (BD Biosciences, Dickinson, Bedford, MA) was coated with fibronectin (25 μg/mL, Sigma-Aldrich) and placed in 24-well plates and engineered hNSCs (1×10⁵ cells/well) were seeded in the upper compartment of the transwell on the next day. After 24 hours of incubation, all lower chambers were washed with PBS, migrated hNSCs were fixed using 3.7% formaldehyde solution (Sigma-Aldrich) for 15 minutes, and the cell underwent permeabilization using 10% coldmethanol (Sigma-Aldrich) for 15 minutes. Crystal violet was used to stain the surface of the transwell for 15 minutes and all transwells were rinsed thoroughly with PBS. Crystal violet stained hNSCs were visualized by fluorescence microscopy (IX-73 Inverted Microscopy, Olympus, Tokyo, Japan) and were counted with Cell Sense Dimension.