Isolating Immune Cells from Mouse Brain and Skull

Summary

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Abstract

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

Introduction

The brain, the central hub of the nervous system, is protected by the skull. Beneath the skull are three layers of connective tissue known as the meninges - the dura mater, arachnoid mater, and pia mater. Cerebrospinal fluid (CSF) circulates in the subarachnoid space between the arachnoid mater and pia mater, cushioning the brain and also removing waste via the glymphatic system^1,2. Together, this unique architecture provides a secure and supportive environment that maintains the stability of the brain and shields it from potential injury.

The brain has long been considered immune-p....

Protocol

The protocol was approved by the Duke Institute Animal Care and Use Committee (IACUC). Male C57Bl/6 mice (3-4 months old; 22-28 g) were used in the current study. The details of the reagents and the equipment used are listed in the Table of Materials.

1. Single-cell suspension from mouse brain

NOTE: Figure 1 illustrates the overview of the brain cell isolation protocol.

1. Anesthetize, intubate.......

Discussion

Here, two simple yet effective protocols are presented for isolating immune cells from the brain and skull bone marrow. These protocols can reliably yield a large quantity of viable immune cells that may be suitable for diverse downstream applications, in particular for flow cytometry.

To study neuroinflammation in various brain disorders, many protocols for immune cell preparations from the brain have been established and used in different laboratories^15,

Materials

Name	Company	Catalog Number	Comments
0.5 mL microcentrifuge tubes	VWR	76332-066
1.5 mL microcentrifuge tubes	VWR	76332-068
15 mL conical tubes	Thermo Fisher Scientific	339651
18 G x 1 in BD PrecisionGlide Needle	BD Biosciences	305195
1x HBSS	Gibco	14175-095
50 mL conical tubes	Thermo Fisher Scientific	339653
96-well V-bottom microplate	SARSTEDT	82.1583
AURORA  flow cytometer	Cytek bioscience
BSA	Fisher	BP9706-100
CD11b-AF594	BioLegend	101254	1:500 dilution
CD19-BV785	BioLegend	115543	1:500 dilution
CD19-FITC	BioLegend	115506	1:500 dilution
CD3-APC	BioLegend	100312	1:500 dilution
CD3-PE	BioLegend	100206	1:500 dilution
CD45-Alex 700	BioLegend	103128	1:500 dilution
CD45-BV421	Biolegend	103133	1:500 dilution
Cell Strainer 70 um	Avantor	732-2758
Dressing Forceps	V. Mueller	NL1410
EDTA	Invitrogen	15575-038
Fc Block	Biolegend	101320	1:100 dilution
Forceps	Roboz	RS-5047
LIVE/DEAD Fixable Blue Dead Cell Stain Kit	Thermo Fisher Scientific	N7167	1:500 dilution
Ly6G-BV421	BioLegend	127628	1:500 dilution
Ly6G-PerCp-cy5.5	BioLegend	127615	1:500 dilution
NK1.1-APC-cy7	BioLegend	108723	1:500 dilution
Percoll (density gradient medium)	Cytiva	17089101
Phosphate buffer saline (10x)	Gibco	70011-044
RBC Lysis Buffer (10x)	BioLegend	420302
Scissors	SKLAR	64-1250
WHEATON Dounce Tissue, 15 mL Size	DWK Life Sciences	357544