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Updated: Jan 14, 2026
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Flow Cytometry and Single-Cell Analysis for Characterizing Microglia Activation in Early Postnatal Mouse Brain Development
Published on: October 3, 2025
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Flow Cytometry and Single-Cell Analysis for Characterizing Microglia Activation in Early Postnatal Mouse Brain
Marianne Mengus1, Benjamin Boucher2, Roqaya Imane2
1Azrieli Research Center of CHU Sainte-Justine; Pediatric Department, Faculty of Medicine, Université de Montréal; marianne.mengus@umontreal.ca.
Journal of Visualized Experiments : Jove
|October 20, 2025
Summary
This study details two methods, flow cytometry and single-cell RNA sequencing, for analyzing microglia in mouse cerebellum. These protocols enable robust investigation of microglial diversity during early brain development and disease.
Area of Science:
- Neuroscience
- Immunology
- Genomics
Background:
- Microglia, the brain's immune cells, have diverse functions influenced by location and condition.
- Understanding microglial heterogeneity is crucial for studying brain development and neurological diseases.
Purpose of the Study:
- To present reproducible protocols for isolating and analyzing microglial populations in mouse cerebellar tissue.
- To enable transcriptomic profiling of microglia for studying developmental changes and disease impacts.
Main Methods:
- Optimized flow cytometry protocol for early postnatal mouse brain tissue, including enzymatic digestion and Percoll gradient centrifugation.
- Single-cell RNA sequencing using 10X Genomics for transcriptomic analysis of isolated microglia.
- Utilized CD45/CD11b gating, live/dead staining, surface marker profiling, and random forest classification for sex identification.
Main Results:
- Established a robust method for high-quality neural cell suspension suitable for microglial analysis.
- Enabled identification of microglial clusters and differential gene expression patterns.
- Validated protocols for reproducible and adaptable microglial research.
Conclusions:
- These complementary methods provide a framework for investigating microglial diversity in the cerebellum.
- The protocols facilitate the study of microglial alterations during early brain development and following experimental perturbations.
