Isolation of Pericytes from Mouse Cortical Tissue Using FACS for Single-cell Sequencing

Summary

To obtain mouse brain cortical pericytes meeting the demands of scRNA-seq, we present a critically optimized FACS protocol based on CD13⁺/CD31⁻ selection. Our key refinements significantly enhance cell viability and RNA integrity, enabling reliable transcriptomic profiling.

Abstract

Pericytes, as a core component of the neurovascular unit, play a vital role in maintaining the integrity of the blood-brain barrier and regulating cerebral hemodynamic homeostasis through modulation of microvascular tone. Dysfunction of pericytes has been implicated in a variety of neurological disorders. To investigate the heterogeneity of pericytes and elucidate their mechanisms in neurovascular diseases at single-cell resolution, we established an optimized and reproducible protocol for isolating mouse brain pericytes suitable for single-cell RNA sequencing (scRNA-seq). This method builds upon the established CD13⁺/CD31^- sorting strategy, but incorporates key enhancements tailored specifically for scRNA-seq applications. Briefly, brains were harvested from three adult C57BL/6 mice. The brain tissues were mechanically dissociated and subjected to enzymatic digestion to obtain single-cell suspensions. After sequential centrifugation, the final cell pellet was resuspended in 20% bovine serum albumin (BSA) solution, a pivotal step we introduced to minimize stress and aggregation, thereby maximizing viability and RNA quality for sequencing. This optimized FACS-based approach thus enables the robust isolation of viable mouse brain pericytes meeting the stringent requirements for scRNA-seq, offering a powerful tool for dissecting pericyte heterogeneity and their pathological roles in neurovascular diseases.

Introduction

Pericytes are an important type of cell located along microvessels, typically embedded within the capillary basement membrane and enveloping endothelial cells¹. In the brain, pericytes, together with endothelial cells, astrocytes, and other cell types, form the neurovascular unit and are integral to the structure and function of the blood-brain barrier (BBB). Pericytes play essential roles in the regulation of cerebral blood flow, vascular remodeling, and maintenance of vascular homeostasis. They maintain the integrity of the BBB through close physical interactions and paracrine signaling with neighboring cells²; dynamic....

Protocol

In this study, we used three adult C57BL/6J mice (3 months old, 20-25 g). All mice were housed under a 12 h light/dark cycle with strictly controlled temperature and humidity, with free access to food and water prior to experiments. All experimental procedures involving laboratory animals were approved by the Laboratory Animal Care and Ethics Committee of the Institute of Microcirculation, Chinese Academy of Medical Sciences, and Peking Union Medical College, and were conducted in strict accordance with relevant animal welfare guidelines.

1. Reagent preparation

1. 30% (wt/vol) Glucose Solution: Dissolve 15 g glucos....

Results

To achieve precise isolation of brain pericytes, cells dissociated from brain tissue were stained with PE Texas Red-conjugated anti-mouse CD41/CD45, FITC-conjugated anti-mouse CD13, and APC-conjugated anti-mouse CD31, establishing a multi-step sorting strategy integrating surface markers and viability detection (Figure 2). Initial gating based on forward scatter (FSC-A) and side scatter (SSC-A) parameters effectively excluded cellular debris (Figure 2, top-left .......

Discussion

This study describes a systematically optimized and validated FACS strategy based on CD13⁺/CD31⁻ multicolor labeling for the highly efficient isolation of mouse brain vascular pericytes²⁴. Flow cytometric and subsequent scRNA-seq analyses confirmed that the method precisely discriminates pericytes from endothelial cells and yields cells that consistently meet all critical quality control metrics for scRNA-seq, including viability, aggregation rate, live cell concentration, and nucleate.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal Bovine Serum (FBS)	Gibicol	A5669701	Aliquot and store at -20°C; can be stored at 4°C for up to 1 week after thawing
Phosphate Buffered Saline(10×PBS, pH 7.4)	Life Technologies	70011-044	Dilute before use for cell washing
D-(+)-Glucose	Sigma-Aldrich	G7021-1KG	For preparing cell energy buffer
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A1933-25g	Protects cells and prevents adhesion
Dulbecco's  Phosphate Buffered Saline (DPBS,1×)	Gibco	14190-144
Hanks' Balanced Salt Solution (HBSS,1×)	Life Technologies	14175-095	For cell washing and resuspension
UltraPure DNase/RNase-Free Distilled Water	Life Technologies	10977-015	For preparing nuclease-free reaction systems
Collagenase/Dispase	Sigma-Aldrich	11097113001-100mg	For tissue digestion and cell dissociation; aliquot and store at -20°C
DNaseI(RNase-Free)	TIANGEN	RT411-1500U	Prevents DNA aggregation; aliquot and store at -20°C
Percoll	Sigma-Aldrich	P1644-100ML	For cell separation; store at 4°C
FITC Rat Anti-Mouse CD13	BD Biosciences	558744	Fluorescent conjugate; store protected from light at 4°C for up to 1 month
APC Rat Anti-Mouse CD31	BD Biosciences	551262	Store protected from light at 4°C for several months
PE Rat Anti-Mouse CD41	BD Biosciences	558040	Store protected from light at 4°C for several months
PE Rat Anti-Mouse CD45	BD Biosciences	553081	Store protected from light at 4°C for several months
FITC Rat IgG1, κ Isotype Control	BD Biosciences	552916	Fluorescence control; store protected from light at 4°C
APC Rat IgG2a, κ Isotype Control	BD Biosciences	553932	Store protected from light at 4°C for several months
PE Rat IgG2b, κ Isotype Control	BD Biosciences	553989	Store protected from light at 4°C for several months
RNase AWAY Decontamination Reagent	Ambion	10328011	For eliminating RNase contamination
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)	BD Pharmingen	553141	Prevent non-specific antibody binding
7-Aminoactinomycin D (7-AAD)	BD Pharmingen	559925	For dead cell exclusion.
2,2,2-Tribromoethanol	Sigma-Aldrich	T48402-25g	For the preparation of 1.25% (w/v) Tribromoethanol Solution
2-Methyl-2-butanol	Psaitong	M20008-500mL	For the preparation of 1.25% (w/v) Tribromoethanol Solution
C57BL/6 mouse	Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.	SYXK (Su) 2020-0041
50 mL centrifuge tube	Corning	430291
15 mL centrifuge tube	Corning	430791
cell cluture dish 100mm×20mm	Corning	430167
Cell Ranger		3.1.0/4.0.0	Single-Cell Sequencing Data Analysis Software
Loupe Cell Browser		4.1	Cell Browser
STAR		2.5.1b	RNA Alignment
irlbpy		-	Principal Component Analysis
tsne		0.15	Barnes-Hut t-SNE (t-Distributed Stochastic Neighbor Embedding) Analysis
k-means		0.17	K-Means Clustering
graph-based clustering		0.17	Graph-Based Clustering
FastQC		v0.11.2	Data Quality Control (QC)