Isolation of human brain endothelial cells and characterization of lipid raft-associated proteins by mass spectroscopy.

The blood-brain barrier (BBB) limits the movements of molecules, nutrients, and cells from the systemic blood circulation into the central nervous system (CNS), and vice versa, thus allowing an optimal microenvironment for CNS development and function. The brain endothelial cells (BECs) form the primary barrier between the blood and the CNS. In addition, pericytes, neurons, and astrocytes that make up the neurovascular unit support the BEC functions and are essential to maintain this restrictive permeability phenotype. To better understand the molecular mechanisms underlying BBB properties, we propose a method to study the proteome of detergent resistant microdomain, namely lipid rafts, from human primary cultures of BECs. This chapter describes a robust human BECs isolation protocol, standard tissue culture protocols, ECs purity assessment protocols, lipid raft microdomain isolation method, and a mass spectrometry analysis technique to characterize the protein content of membrane microdomains.