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FACS isolation of endothelial cells and pericytes from mouse brain microregions

Nature Protocols volume 13, pages 738–751 (2018)Cite this article

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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.

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Figure 1: Overview of protocol for purification of brain endothelial cells and pericytes by FACS.
Figure 2: CD13 is a specific pericyte marker in the adult mouse brain.
Figure 3: Validation of FACS-purified endothelial cells and pericytes.
Figure 4: Potential applications of FACS-purified primary endothelial cells and pericytes.
Figure 5: Schema outlining splitting and pooling of samples at key steps in the protocol.
Figure 6: Representative FACS plots showing the gating strategy for the purification of endothelial cells and pericytes from the adult mouse brain.
Figure 7: Morphology and immunostaining of cultured endothelial cells and pericytes.

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Acknowledgements

This work was supported by NIH NINDS R21NS075610, NIH NINDS R01NS074039, and NYSTEM C026401 (F.D.); the Leona M. and Harry B. Helmsley Charitable Trust (F.D.); The David and Lucile Packard Foundation (F.D.); and NIH NICHD T32HD055165 and NIH NIGMS 5T32GM007367 (E.E.C.). This work was also supported by the Jerry and Emily Spiegel Laboratory for Cell Replacement Therapies and the University of Basel. We thank C.-H. Liu and K. Gordon from the Herbert Irving Comprehensive Cancer Center of Columbia University for assistance with FACS and flow cytometry, C. Segalada for comments on the manuscript, and V. Silva-Vargas and A. Delgado for comments on the manuscript and assistance with preparation of the figures.

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Authors and Affiliations

  1. Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA

    Elizabeth E Crouch

  2. Biozentrum, University of Basel, Basel, Switzerland

    Fiona Doetsch

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  1. Elizabeth E Crouch
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E.E.C. designed the research, performed research, analyzed data, and wrote the paper. F.D. designed the research, analyzed data, and wrote the paper.

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Correspondence to Fiona Doetsch.

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Integrated supplementary information

Supplementary Figure 1 Effect of different enzymes on CD13 and CD31 populations.

Comparison of cortex dissociated for 30 minutes with collagenase/dispase digestion (a) or papain (b). Papain degrades the CD31 epitope and CD13+ pericytes are greatly reduced (b). Percentages refer to the proportion of cells in the previous parent gate. Plots show 50,000 events. Experiments were performed with the approval of Columbia University IACUC and the cantonal veterinary office Basel-Stadt.

Supplementary Figure 2 FACS controls for setting gating strategy.

Representative plots for isotype, single colour and FMO controls for FACS strategy. Isotype controls and the percentage of non-specific labeling for each fluorophore are shown in a (FITC), b (PE), and c (APC). Unstained cells (d-e), single colour controls: CD45-PE and CD41-PE (f); CD31-APC (g); CD13-FITC (h) and FMO controls (CD13-FITC (i); CD31-APC (j)) are used to set gating strategy. Plots show 50,000 events. Experiments were performed with the approval of Columbia University IACUC and the cantonal veterinary office Basel-Stadt. a–c adapted with permission from Crouch et al. (ref. 21), Regional and stage-specific effects of prospectively purified vascular cells on the adult V-SVZ neural stem cell lineage, J. Neurosci., vol. 35, Copyright 2015; permission conveyed through Copyright Clearance Center.

Supplementary Figure 3 Schema of dissection of cortex.

Overview of dissection of cortex from the brain. Steps refer to numbers in Box 1. First, remove the meninges from brain surface. Make four coronal cuts in the brain. Dissect the cortex from each coronal section. Use the corpus callosum (black) as a ventral limit to dissect the cortex (dashed white line).

Supplementary Figure 4 Dissection of the mouse cortex.

(a-b) Dorsal view of whole mouse brain before (a) and after (b) removal of the meninges. (c-d) Coronal slice of the mouse brain before (c) and after (d) dissecting the cortex. Experiments were performed with the approval of Columbia University IACUC and the cantonal veterinary office Basel-Stadt.

Supplementary Figure 5 Schema of dissection of V-SVZ tissue.

Overview of V-SVZ dissection. Steps refer to numbers in Box 2. To dissect the V-SVZ from the brain, make 4 coronal cuts through the brain and discard the most rostral and caudal sections. From each coronal section, dissect the V-SVZ (thin brown wedge beside the lateral ventricle) by cutting ventrally below the ventricle (step 3), dorsally by pulling up on the corpus callosum (step 4), and laterally to remove striatum (step 5). Repeat on the contralateral side before proceeding to the next slice.

Supplementary Figure 6 Dissection of the mouse V-SVZ.

(a) Photograph of coronal section of mouse brain showing bilateral V-SVZ outlined by dashed lines. (b-c) The V-SVZ has a distinct, more velvety texture than the adjacent striatum, which contains white matter tracts. The V-SVZ is located to the right of the dashed line in (c) adjacent to the lateral ventricle. Cc, corpus callosum; str, striatum. Experiments were performed with the approval of Columbia University IACUC and the cantonal veterinary office Basel-Stadt.

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Crouch, E., Doetsch, F. FACS isolation of endothelial cells and pericytes from mouse brain microregions. Nat Protoc 13, 738–751 (2018). https://doi.org/10.1038/nprot.2017.158

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