Labeling Neural Cell Surface Markers with Azidosugar while Co-culturing with Endothelial Cells

Begin by adding a mouse brain endothelial cell suspension to the permeable membrane insert of a transwell plate.

Add a growth medium to the bottom well and incubate.

The growth factors and nutrients in the medium help the cells to grow as a monolayer.

Remove the medium from the bottom well and from the insert.

Wash the insert's inner and outer surfaces to remove any debris.

Add an adherent medium to the insert, and transfer the insert to a matrix-coated well containing primary cortical neural stem and progenitor cells, or NSPCs, and incubate.

Add azidosugar, an unnatural sialic acid precursor analog, to both the endothelial cells and the NSPCs, and incubate.

The cytokines and chemokines secreted by endothelial cells promote NSPCs' growth into large sheet-like clones.

Azidosugars integrate into the sialoglycan structures on the NSPC cell surface, labeling the glycoprotein. This enables the identification of cell surface proteins on the expanded NSPCs.

To prepare the endothelial cells, resuspend a BEND3 cell pellet from a 0.10-centimeter petri dish at 90% confluence with 9 milliliters of BEND3 cell medium. Add 1 milliliter of the cell suspension into one permeable support insert. Add