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Bio Protoc. 2018 Nov 20;8(22). pii: e3092. doi: 10.21769/BioProtoc.3092.

Collagenase-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry.

Author information

1
Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
2
Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Stanford, CA, USA.
3
VA Palo Alto Health Care System, Palo Alto, CA, USA.
4
Stanford Neurosciences Institute, Stanford University, Stanford, CA, USA.
5
Palo Alto Veterans Institute for Research, Palo Alto, CA 94304, USA.

Abstract

The brain endothelium is a highly specialized vascular structure that maintains the activity and integrity of the central nervous system (CNS). Previous studies have reported that the integrity of the brain endothelium is compromised in a plethora of neuropathologies. Therefore, it is of particular interest to establish a method that enables researchers to investigate and understand the molecular changes in CNS endothelial cells and underlying mechanisms in conjunction with murine models of disease. In the past, approaches to isolate endothelial cells have either involved the use of transgenic reporter mice or suffered from insufficiently pure cell populations and poor yield. This protocol here is based on well-established protocols that were modified and combined to allow single cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after careful removal of the meninges and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested in two steps resulting in a single cell suspension. Cells are stained with a cocktail of fluorochrome-conjugated antibodies identifying not only brain endothelial cells, but also potentially contaminating cell types such as pericytes, astrocytes, and lineage cells. Using flow cytometry, cell populations are separated and sorted directly into either RNA lysis buffer for bulk RNA analyses (e.g., RNA microarray and RNA-Seq) or in pure fetal bovine serum to preserve viability for other downstream applications such as single cell RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq). The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 3 × 105 cells from a pool of four adult mice.

KEYWORDS:

Blood-brain barrier; Brain endothelium; CNS; Neuro-vascular unit; Pericytes; RNA microarray; RNA-Seq; Single cell isolation

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