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Updated: Aug 28, 2025
Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
Using mechanical homogenization to isolate microglia from mouse brain tissue to preserve transcriptomic integrity
Shawn Herron1, Jean-Christophe Delpech2, Charlotte Madore2
1Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA, USA.
Abstract:
Numerous approaches have been developed to isolate microglia from the brain, but procedures using enzymatic dissociation at 37°C can introduce drastic transcriptomic changes and confound results from gene expression assays. Here, we present an optimized protocol for microglia isolation using mechanical homogenization. We use Dounce homogenization to homogenize mouse brain tissue into single-cell suspension. We then isolate microglia through Percoll gradient and flow cytometry. Isolated microglia exhibit a gene expression pattern without the changes induced by heated enzymatic digestion. For complete details on the use and execution of this protocol, please refer to Clayton et al. (2021).
Insights
This study introduces a new method for isolating microglia, a type of brain cell, using mechanical homogenization instead of heat and enzymes. This preserves the cells' natural gene expression for more accurate research.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia isolation is crucial for studying brain health and disease.
- Current enzymatic methods at 37°C can alter microglia gene expression, affecting research outcomes.
- Accurate microglia transcriptomics are essential for understanding neurological conditions.
Purpose of the Study:
- To develop an optimized protocol for isolating microglia with minimal transcriptomic changes.
- To provide a reliable method for obtaining high-quality microglia for gene expression analysis.
- To overcome the limitations of enzymatic dissociation in microglia research.
Main Methods:
- Mechanical homogenization of mouse brain tissue using Dounce homogenizer.
- Single-cell suspension preparation.
- Microglia isolation via Percoll gradient and flow cytometry.
Main Results:
- The optimized protocol successfully isolates microglia.
- Isolated microglia show preserved gene expression patterns, avoiding heat-induced artifacts.
- Mechanical homogenization prevents transcriptomic alterations common in enzymatic methods.
Conclusions:
- This mechanical homogenization protocol offers a superior alternative for microglia isolation.
- The method ensures the integrity of microglia gene expression for reliable downstream assays.
- This technique is vital for advancing research in neuroinflammation and brain disorders.
