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

1. Preparation of Mouse Endothelial Culture in Permeable Support Inserts

NOTE: BEND3 cells are maintained according to the manufacturer's instructions.

1. Prepare BEND3 cell medium (BM) by adding 50 mL of FBS and 5 mL of penicillin-streptomycin into 500 mL of DMEM and mix well.
2. Aspirate the medium from the dish and wash the BEND3 cell culture with 1 mL of PBS once. Add 1 mL of 0.25% trypsin-EDTA (ethylene diamine tetraacetic acid) to the cells and incubate them for 4 min at 37 °C.
3. Add 1 mL of BM to the cells to neutralize trypsin-EDTA and gently pipette up and down to completely dissociate the cells. Transfer the cell suspension into a new 15 mL conical tube and pellet by centrifugation at room temperature (RT) for 5 min at 400 x g.
4. Aspirate the supernatant from the tube and resuspend the cells with 9 mL of fresh BM, then add 1 mL of cell suspension into one permeable support insert. Add another 2 mL of fresh BM per well at the bottom chamber of the matrix. Continue to culture the cells for one day.

2. Set-up of Neural-endothelial Co-culture and Ac4ManNAz Labeling System

1. One day, after plating BEND3 cells in the inserts, gently aspirate the medium in the bottom chamber first, then the inserts. Wash the enface of the inserts 3 times with pre-warmed DMEM (Dulbecco's modified eagle medium). Wash the outer surface of the inserts by rinsing with pre-warmed DMEM.
2. Add 1 mL of pre-warmed AM (adherent medium) into one insert, then transfer the inserts into the wells with primary cortical cells. Incubate the co-culture at 37 °C and 5% CO₂ for 12 h.
3. Dissolve Ac4ManNAz in DMSO (dimethyl sulfoxide) to achieve a stock concentration of 200 mM. 12 h after setting up the neural-endothelial co-culture, add 1 µL of Ac4ManNAz stock per bottom chamber and 0.5 µL of stock per insert into the co-culture. Shake the plates immediately and gently to mix the medium well. For the control cells, add an equal volume of DMSO.
4. Culture the cells for another 5 days at 37 °C and 5% CO₂. Prepare the AM with 10x bFGF as refeeding medium (RM). During this time, add 100 µL of RM per insert and 200 µL of RM per bottom chamber to refeed the endothelial and neural cells every other day. During the refeeding, do not supply Ac4ManNAz or DMSO into the culture.