High-yield isolation of murine microglia by mild trypsinization
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Updated: Aug 30, 2026
Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation
Published on: February 16, 2018
Josep Saura1, Josep Maria Tusell, Joan Serratosa
1Department of Pharmacology and Toxicology, Institut d'Investigacions Biomèdiques de Barcelona, IIBB-CSIC, Barcelona, Spain. jsafat@iibb.csic.es
Abstract:
Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05-0.12%) in the presence of 0.2-0.5 mM EDTA and 0.5-0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as >98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon gamma, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-kappaB translocation, NO, and tumor necrosis alpha release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.
Insights
A new method isolates highly pure microglia from mouse pups with over five times the yield of traditional techniques. This improved microglial isolation method offers reproducibility and convenience for research applications.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia isolation is crucial for studying neuroinflammation and brain function.
- Traditional methods yield low numbers of microglia, limiting research scope.
- High-purity microglial cultures are essential for reliable experimental outcomes.
Purpose of the Study:
- To develop a novel, high-yield method for isolating pure microglial cultures.
- To improve upon existing techniques for microglial isolation from mixed glial cultures.
- To provide a reproducible and efficient method for obtaining substantial quantities of microglia.
Main Methods:
- Utilizing mild trypsinization (0.05-0.12%) with EDTA and Ca2+ on mixed glial cultures.
- Detaching astrocytes while leaving firmly attached microglia undisturbed.
- Culturing isolated microglia for extended periods to assess viability and function.
Main Results:
- Achieved an average yield of ~2,000,000 microglial cells/mouse pup, a >5-fold increase.
- Isolated microglial preparations demonstrated >98% purity.
- Cultured microglia exhibited proliferation, phagocytosis, and typical responses to immune stimuli (M-CSF, LPS, IFN-γ).
Conclusions:
- The new method provides high-purity, high-yield microglial cultures with ease and reproducibility.
- This technique is particularly beneficial for studies requiring large amounts of microglial protein/mRNA.
- The method is suitable for microglial isolation from limited starting materials, including transgenic animals.
