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Isolation and flow cytometric characterization of newborn mouse brain-derived microglia maintained in vitro
N F Hassan1, S Rifat, D E Campbell
1Division of Infectious Diseases and Immunology, Joseph Stokes, Jr., Research Institute, Children's Hospital of Philadelphia, PA 19104.
Abstract:
Microglia have been identified in the white matter of developing and adult mouse brain using different murine macrophage markers. While several techniques for the isolation of murine microglia have been described, the small cell yields and partial purification have limited the progress of these studies. We now describe the isolation of murine microglia using a modification of McCarthy and de Vellis method. Brain tissues from 1-2 day old newborn mice were mechanically and chemically dissociated and maintained in in vitro culture for 3 weeks. In primary dissociated brain cultures, microglia are observed after 10 days migrating from small colonies. After 16-20 days, brain-derived microglia were isolated with high cell yields by continuous shaking of the cultures for 16 hr. In contrast to resident murine peritoneal macrophages, microglia express less Class II (Ia) antigen and a small percentage express L3T4 (CD4) antigen by flow cytometry.
Insights
Researchers developed an improved method for isolating murine microglia, achieving high cell yields. This technique enhances the study of these crucial brain immune cells and their functions.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglia, the resident immune cells of the brain, are critical for central nervous system development and function.
- Existing methods for isolating murine microglia often result in low cell yields and incomplete purification, hindering research.
- Understanding microglia requires efficient and reliable isolation techniques.
Purpose of the Study:
- To describe a modified method for the isolation of murine microglia with high cell yields.
- To improve upon the McCarthy and de Vellis method for microglia isolation.
- To facilitate further research into microglia biology and function.
Main Methods:
- Mechanical and chemical dissociation of newborn mouse brain tissue.
- In vitro culture of dissociated cells for up to 3 weeks.
- Isolation of microglia via continuous shaking of cultures after 16-20 days.
Main Results:
- Microglia were observed migrating from colonies in primary dissociated brain cultures after 10 days.
- High cell yields of brain-derived microglia were obtained using the modified isolation method.
- Flow cytometry revealed that isolated microglia express less Class II (Ia) antigen and a small percentage express L3T4 (CD4) antigen compared to peritoneal macrophages.
Conclusions:
- The described modified method provides an efficient means to isolate murine microglia with high yields.
- This improved isolation technique will support advanced studies on microglia in the mouse brain.
- Characterization of isolated microglia reveals distinct antigen expression profiles compared to peripheral macrophages.