FACS isolation of endothelial cells and pericytes from mouse brain microregions

Abstract

The vasculature is emerging as a key contributor to brain function during neurodevelopment and in mature physiological and pathological states. The brain vasculature itself also exhibits regional heterogeneity, highlighting the need to develop approaches for purifying cells from different microregions. Previous approaches for isolation of endothelial cells and pericytes have predominantly required transgenic mice and large amounts of tissue, and have resulted in impure populations. In addition, the prospective purification of brain pericytes has been complicated by the fact that widely used pericyte markers are also expressed by other cell types in the brain. Here, we describe the detailed procedures for simultaneous isolation of pure populations of endothelial cells and pericytes directly from adult mouse brain microregions using fluorescence-activated cell sorting (FACS) with antibodies against CD31 (endothelial cells) and CD13 (pericytes). This protocol is scalable and takes ∼5 h, including microdissection of the region of interest, enzymatic tissue dissociation, immunostaining, and FACS. This protocol allows the isolation of brain vascular cells from any mouse strain under diverse conditions; these cells can be used for multiple downstream applications, including in vitro and in vivo experiments, and transcriptomic, proteomic, metabolomic, epigenomic, and single-cell analysis.

Disruption of the Blood-Brain Barrier in 22q11.2 Deletion Syndrome

“…Microvascular endothelial cells were isolated from the anterior portion of the CNS for flow cytometry analysis as previously described (Crouch & Doetsch, 2018 analyses were performed blinded and using ImageJ, as previously described (Alvarez et al, 2015).…”

A systems-based map of human brain cell-type enriched genes and malignancy-associated endothelial changes

“…PDGFRB, ACTA2, DES, MCAM (Smyth et al, 2018), VTN and IFITM1 (He et al, 2016), were not classified as EC-enriched. ANPEP (CD13), a marker often used to identify pericytes in mouse (Crouch and Doetsch, 2018), was classified as brain EC-enriched in our study, however, it was also classified as such by scRNAseq (Darmanis et al, 2015) and is highly expressed in EC lines (Thul et al, 2017). Thus, whilst our data indicates that pericyte genes are not generally incorrectly classified as EC-enriched, we cannot definitively exclude the possibility that the EC-enriched list contains dual pericyte/EC-enriched transcripts.…”

High expression levels of CLEC4M indicate poor prognosis in patients with hepatocellular carcinoma

“…The expression level of CLEC4M in HCC cells was also investigated. CD31 is a biomarker that is commonly used to label endothelial cells (27). Thus, co-localization staining, with CLEC4M and CD31 antibodies, was performed using the pathological tissue sections.…”

“…Additionally, in tissues containing many endothelial cells, the staining of CLEC4M appears stronger. Liver sinusoids consist of a line of sinusoidal endothelial liver cells and Kupffer cells, providing oxygen and nutrients to hepatocytes and forming a distribution network throughout the liver (27,35,36). Additionally, CLEC4M can bind to intercellular adhesion molecule 3 (ICAM3; 28), resulting in the activation and recruitment of ICAM3-positive T cells and initiating an immune response to pathogens or cancer cells (37).…”