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 another 2 milliliters of BEND3 medium per well at the bottom chamber of the matrix. Continue to culture the cells for one day at 37 degrees Celsius with 5% carbon dioxide.

One day after plating the cells in the inserts, gently aspirate the medium, first from the bottom chamber and then from the inserts. Wash the face of the inserts three times with pre-warmed DMEM. Wash the outer surface of the inserts by rinsing with pre-warmed DMEM. Next, add 1 milliliter of pre-warmed AM into one insert. Transfer the inserts into the wells with primary cortical cells. Incubate the co-culture at 37 degrees Celsius with 5% carbon dioxide for 12 hours.

Dissolve Ac₄ManNAz in DMSO to achieve a stock concentration of 200 millimolar. Retrieve the neural endothelial co-culture plate from the incubator. Add 1 microliter of the Ac₄ManNAz stock to each bottom chamber and 0.5 microliters per insert into the co-culture. Culture the cells at 37 degrees Celsius with 5% carbon dioxide for 5 days. While the cells are culturing, add 100 microliters of RM per insert and 200 microliters of RM per bottom chamber to refeed the endothelial and neural cells every other day.