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

Abstract

Source: Bai, Q., et al. Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan. J. Vis. Exp. (2019).

This video demonstrates a protocol for co-culturing mouse brain endothelial cells and primary cortical stem cells and labeling the cell surface glycoproteins using Ac4ManNAz (azidosugar per-O-acetylated N-azidoacetylmannosamine). This method allows for selective enrichment and identification of cell surface proteins in primary cells.

Protocol

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 ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BEND3	ATCC	CRL-229
DMEM	Gibco	11960044
L-glutamine	Gibco	25030081	1%
Sodium pyruvate	Sigma	P5280	1%
N2 supplement	Gibco	17502048	1 to 100
N-acetyl-L-cysteine	Sigma	A7250	1 mM
B27 supplement	Gibco	17504044	1 to 50
Poly-L-lysine	Sigma	P4707
basic Fibroblast growth factor	Gibco	PHG0261	10 ng/mL
Penicillin-Streptomycin	Gibco	15140122	1%
Fetal bovine serum	Gibco	10099141	10%
HBSS	Gibco	14175095
Tripsin-EDTA, 0.25%	Gibco	25200056
DPBS	Gibco	14190094
Transwell	Corning	3450