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

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.