Cold methanol

To examine whether engineered hNSCs can migrate to lymph node–derived metastatic colorectal adenocarcinoma, SW-620 cells (1×10⁵ cells per well) and human dermal fibroblast cells (a control, 1×10⁵ cells per well) were plated in a 24-well plate containing 1% CD-FBS phenol free DMEM medium with incubation for 24 hours. The bottom surface of transwell plates (0.4 μm, BD Biosciences, Dickinson, Franklin Lakes, NJ) coated the with fibronectin (250 μg/mL, Sigma-Aldrich) was placed in the 24-well plates and CM-Dil (Invitrogen Life Technologies) pre-stained engineered hNSCs were plated in the upper chambers of the transwell plates at a density of 1×10⁵ cells per well in the same condition medium and cultured for 24 hours at 37°C. After washing the lower chamber, the cells were fixed with 10% formalin solution (Sigma-Aldrich) for 10 minutes and permeabilized using 100% cold-methanol (Sigma-Aldrich) for 10 minutes. Then, DAPI (4',6-diamidino-2-phenylindole, Invitrogen Life Technologies) was added to the lower chamber at 300 nM and the plates were incubated for 10 minutes at 37°C followed by washing with PBS. Cells stained with CM-Dil and DAPI were examined by fluorescence microscopy (IX-73 Inverted Microscopy, Olympus, Tokyo, Japan) and counted by Cell Sense Dimension.

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.