Brain endothelial cells are the major building block of the blood-brain barrier. To study the
role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here,
we describe a protocol in which vessel fragments are isolated from adult mice. After density
centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected
by puromycin treatment and grown to confluence within one week.