Related Experiment Video
Updated: Jul 12, 2025
Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing
Published on: December 3, 2019
A simple protocol for isolating microglia from adult mouse brain
Sudipta Chakrabarti1, Sukhamoy Gorai1, Kalipada Pahan1,2
1Department of Neurological Sciences, Rush University Medical Center, Chicago, USA.
Objectives:
Although microglia are activated in adult and aged brains resulting in neurodegenerative and neuroinflammatory disorders, most of the cell culture studies on microglia deal with neonatal microglia because of ease of isolation. Microglia could be isolated from adult brains, but it requires separation by density gradient centrifugation, magnetic beads, etc. Here, we describe a simple protocol of isolating highly purified microglia from adult mouse brains.
Methods:
Our protocol involves dilution with sterile PBS or media, regular centrifugation, and plating on poly-D-lysine-coated flasks.
Results:
These adult microglia expressed the inducible nitric oxide synthase in response to preformed α-syn fibril, an etiological reagent of Parkinson's disease, and bacterial lipopolysaccharides, one of the prototype proinflammatory stimuli. Moreover, these adult microglia exhibited phagocytosis, which was stimulated by LPS treatment.
Conclusions:
These results suggest that adult microglia isolated by our procedure are functional and that these adult microglia could be used for studies related to neurodegenerative disorders.
Insights
We developed a simple method to isolate functional adult microglia from mouse brains. This technique enables research into neurodegenerative diseases using adult microglia, overcoming limitations of neonatal cell cultures.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia activation is implicated in neurodegenerative and neuroinflammatory disorders.
- Neonatal microglia are commonly used in cell culture due to ease of isolation.
- Adult microglia isolation typically requires complex methods like density gradient centrifugation or magnetic beads.
Purpose of the Study:
- To present a straightforward protocol for isolating highly purified microglia from adult mouse brains.
- To demonstrate the functionality of adult microglia obtained through the described method.
Main Methods:
- Dilution of adult mouse brain tissue with sterile PBS or media.
- Standard centrifugation steps.
- Plating of isolated cells on poly-D-lysine-coated flasks.
Main Results:
- Isolated adult microglia expressed inducible nitric oxide synthase (iNOS) in response to alpha-synuclein fibrils (Parkinson's disease reagent) and lipopolysaccharides (LPS).
- Adult microglia demonstrated phagocytic activity, which was enhanced by LPS treatment.
Conclusions:
- The described protocol yields functional adult microglia.
- These isolated adult microglia are suitable for research on neurodegenerative disorders.
