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Isolating Immune Cells from Mouse Brain and Skull
In This Article
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 system1,2. 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.
- Anesthetize, intubate.......
Representative Results
To prepare immune cells from the mouse brain tissue, the protocol generally yields cells with high viability (84.1% ± 2.3% [mean ± SD]). Approximately 70%-80% of these cells are CD45 positive. In the normal mouse brain, nearly all CD45+ cells are microglia (CD45LowCD11b+), as expected. This protocol has been used in the laboratory for various applications, including flow cytometry analysis, fluorescence-activated cell sorting (FACS), and scRNA-seq analysis. As an examp.......
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 laboratories15,
Acknowledgements
We thank Kathy Gage for her excellent editorial contribution. The illustration figures were created with BioRender.com. This study was supported by funds from the Department of Anesthesiology (Duke University Medical Center) and NIH grants NS099590, HL157354, and NS127163.
....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 |
References
- Bohr, T., et al. The glymphatic system: Current understanding and modeling. iScience. 25 (9), 104987 (2022).
- Jiang-Xie, L. F., et al. Neuronal dynamics direct cerebrospinal fluid perfusion and brain clearance.
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