Background

Endothelial cells in the brain form a specialized barrier that limits and controls the passage of large and small molecules from the bloodstream into the brain (Zlokovic, 2008). In their natural niche, brain endothelial cells interact closely with several cell types, including pericytes, glia, and immune cells, which are integral to brain homeostasis (Armulik et al., 2005; Gavins et al., 2007). The study of brain endothelial cell biology has benefited greatly from the use of procedures to isolate either endothelium or vascular complexes, which include endothelial cells, pericytes, and glial contacts (radial glia, oligodendrocytes, and astrocyte endfeet). The former is normally achieved by fluorescence-activated cell sorting (FACS) isolation (Daneman et al., 2010), whereas the latter is done by centrifugation in a dextran solution (Yousif et al., 2007). FACS-based isolation can alter the properties of endothelial cells and is expensive and inefficient, leading to the isolation of very few cells that may not reflect their native state. These issues are more evident when obtaining samples from embryonic brains, where the sample is extremely limited, and fewer than 5000 endothelial cells are typically obtained from one brain. This number is adequate for transcriptional analyses using specialized kits (Santander et al., 2020) but is insufficient for biochemical assays. Conversely, dextran-based isolation of vascular complexes has not been applied to embryonic brains because the standard protocol requires a much bigger sample obtained from single mouse embryos. The ability to obtain enough sample for biochemical assays from single embryonic brains is necessary for the study of developmental vascular diseases, such as the Fowler syndrome (Santander et al., 2020). Here, to overcome these sample size issues, we describe the adaptation of the paradigm of dextran-based isolation of vascular fragments to individual mouse embryo brains. We demonstrate the utility of vascular fragment isolation to measure cholesterol content in embryonic brain blood vessels, an assay not feasible using FACS-isolated endothelial cells.