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Isolation of Cortical Microglia with Preserved Immunophenotype and Functionality From Murine Neonates
Published on: January 30, 2014
Efficient isolation of live microglia with preserved phenotypes from adult mouse brain
Maria Nikodemova1, Jyoti J Watters
1Department of Comparative Biosciences, University of Wisconsin, Madison, WI 53706, USA.
Background:
Microglial activation plays a key role in the neuroinflammation associated with virtually all CNS disorders, although their role in normal CNS physiology is becoming increasingly appreciated. Neuroinflammation is often assessed by analyzing pro-inflammatory mediators in CNS tissue homogenates, under the assumption that microglia are the main source of these molecules. However, other cell types in the CNS can also synthesize inflammatory molecules. Hence, to enable direct analysis of microglial activities ex vivo, an efficient, reliable, and reproducible method of microglial isolation is needed.
Methods:
After enzymatic digestion of brain tissues and myelin removal, CD11b⁺ cells were isolated using immunomagnetic separation, yielding highly purified microglia without astrocyte or neuronal contamination. We used three methods of myelin removal (30% Percoll, 0.9 mol/l sucrose and anti-myelin magnetic beads), and compared their effects on microglial viability and yield. To determine whether the isolation procedure itself activates microglia, we used flow cytometry to examine microglial properties in brain-tissue homogenates and isolated microglia from control and lipopolysaccharide (LPS) -treated mice.
Results:
This method yielded a highly purified CD11b⁺ cell population with properties that reflected their in vivo phenotype. The viability and yield of isolated cells were significantly affected by the myelin removal method. Although the microglial phenotype was comparable in all methods used, the highest viability and number of CD11b⁺ cells was obtained with Percoll. Microglia isolated from LPS-treated mice displayed a pro-inflammatory phenotype as determined by upregulated levels of TNF-α, whereas microglia isolated from control mice did not.
Conclusions:
Immunomagnetic separation is an efficient method to isolate microglia from the CNS, and is equally suitable for isolating quiescent and activated microglia. This technique allows evaluation of microglial activities ex vivo, which accurately reflects their activities in vivo. Microglia obtained by this method can be used for multiple downstream applications including qRT-PCR, ELISA, Western blotting, and flow cytometry to analyze microglial activities in any number of CNS pathologies or injuries.
Insights
A new immunomagnetic separation method efficiently isolates pure microglia from the central nervous system (CNS). This technique accurately reflects in vivo microglial activity for studying neuroinflammation and CNS disorders.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Microglial activation is central to neuroinflammation in CNS disorders.
- Assessing neuroinflammation often relies on analyzing mediators in CNS homogenates, assuming microglia are the sole source.
- Other CNS cell types can also produce inflammatory molecules, necessitating direct microglial analysis.
Purpose of the Study:
- To develop an efficient, reliable, and reproducible method for isolating microglia from CNS tissue.
- To enable direct ex vivo analysis of microglial activities.
- To compare different myelin removal techniques for optimal microglial isolation.
Main Methods:
- Enzymatic digestion of brain tissue followed by myelin removal.
- Isolation of CD11b+ cells using immunomagnetic separation.
- Comparison of three myelin removal methods (Percoll, sucrose, anti-myelin beads) for yield and viability.
- Flow cytometry analysis to assess microglial properties and activation status.
Main Results:
- Immunomagnetic separation yielded highly purified microglia (CD11b+) without astrocyte or neuronal contamination.
- Percoll resulted in the highest viability and yield of isolated microglia.
- Isolated microglia from lipopolysaccharide (LPS)-treated mice showed an upregulated pro-inflammatory phenotype (TNF-α), reflecting in vivo activation.
- Microglia from control mice maintained a quiescent phenotype.
Conclusions:
- Immunomagnetic separation is an effective method for isolating both quiescent and activated microglia from the CNS.
- The isolation procedure accurately reflects in vivo microglial activities.
- Isolated microglia are suitable for various downstream applications (qRT-PCR, ELISA, Western blotting, flow cytometry) to study CNS pathologies.
