FACS Isolation of Endothelial Cells and Pericytes From Mouse Brain Microregions

Nat Protoc. 2018 Apr;13(4):738-751. doi: 10.1038/nprot.2017.158. Epub 2018 Mar 22.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / metabolism
  • Brain / cytology*
  • CD13 Antigens / metabolism
  • Endothelial Cells / physiology*
  • Flow Cytometry / methods*
  • Fluorescent Antibody Technique
  • Mice
  • Pericytes / physiology*
  • Platelet Endothelial Cell Adhesion Molecule-1 / metabolism
  • Staining and Labeling / methods

Substances

  • Antibodies
  • Pecam1 protein, mouse
  • Platelet Endothelial Cell Adhesion Molecule-1
  • CD13 Antigens