Efficient isolation of live microglia with preserved phenotypes from adult mouse brain

Maria Nikodemova,Jyoti J Watters

doi:10.1186/1742-2094-9-147

Abstract

BackgroundMicroglial 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.MethodsAfter 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.ResultsThis 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.ConclusionsImmunomagnetic 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.

Highlights

Emerging evidence indicates the active involvement of microglia, Central nervous system (CNS) resident innate immune cells, in virtually all aspects of physiology in the healthy, diseased, and injured CNS [1,2,3]
Neuroinflammation is often assessed by analyzing cytokine expression in CNS tissue homogenates; this method lacks cell specificity even though microglia are usually presumed to be the source of inflammatory molecule production in these samples
We found that cell viability was highest using Percoll, followed by anti-myelin beads, as determined by either Trypan blue exclusion (Table 2) or cell staining with the Live/Dead stain (Figure 2)

Summary

Introduction

Emerging evidence indicates the active involvement of microglia, CNS resident innate immune cells, in virtually all aspects of physiology in the healthy, diseased, and injured CNS [1,2,3]. Neuroinflammation is often assessed by analyzing cytokine expression in CNS tissue homogenates; this method lacks cell specificity even though microglia are usually presumed to be the source of inflammatory molecule production in these samples. An efficient, reliable and highly reproducible method of microglial isolation is needed to allow direct analysis of their properties ex vivo, regardless of their phenotype 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. 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. 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

Methods

Results

Discussion

Conclusion