Protocol for isolation of endothelial cells from adult mouse brain

Brain endothelial cells (ECs) line blood vessel walls and form the blood-brain barrier (BBB), facilitating interactions between the brain and the periphery. Here, we present a protocol for isolating purified ECs from adult mouse brains using fluorescence-activated cell sorting (FACS) for downstream analyses, such as RNA sequencing. We describe the steps for brain dissection, enzymatic tissue digestion, trituration, myelin removal, and FACS. Furthermore, we detail adaptations of this protocol for isolating ECs from brain subregions, such as the hippocampus. For complete details on the use and execution of this protocol, please refer to Munji et al.¹ and Pulido et al.².