Abstract
In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, the role of each central nervous system (CNS)-resident cell type during inflammation, neurodegeneration, and remission has been frequently addressed. Although protocols for the isolation of different individual CNS-resident cell types exist, none can harvest all of them within a single experiment. In addition, isolation of individual cells is more demanding in adult mice and even more so from the inflamed CNS. Here, we present a protocol for the simultaneous purification of viable single-cell suspensions of all principal CNS-resident cell types (microglia, oligodendrocytes, astrocytes, and neurons) from adult mice—applicable in healthy mice as well as in EAE. After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes and, neurons were isolated via magnetic-activated cell sorting (MACS). Validations comprised flow cytometry, immunocytochemistry, as well as functional analyses (immunoassay and Sholl analysis). The purity of each cell isolation averaged 90%. All cells displayed cell-type-specific morphologies and expressed specific surface markers. In conclusion, this new protocol for the simultaneous isolation of all major CNS-resident cell types from one CNS offers a sophisticated and comprehensive way to investigate complex cellular networks ex vivo and simultaneously reduce mice numbers to be sacrificed.
Highlights
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system via demyelination accompanied by axonal damage and neurodegeneration
After dissociation of the brain and spinal cord from adult mice, microglia, oligodendrocytes, astrocytes, and neurons were isolated via magnetic-activated cell sorting MACS [35]
Our analysis showed that while neutrophils expressed high levels of both Ly6C and Ly6G, Ly6 expression by the isolated microglia fraction was almost congruent with the Fluorescence Minus One (FMO) (Table 5, Supplemental Figure S1f,g)
Summary
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease affecting the central nervous system via demyelination accompanied by axonal damage and neurodegeneration. Over the last years, many cutting-edge protocols have been developed for the differentiation of these cell types from neural stem cells or progenitors, which have enabled the detailed study of molecular mechanisms in vitro [14,29,30,31,32,33,34]. These protocols cannot sufficiently address complex intercellular networks and can only be investigated in an in vivo system
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