Primary microglia, in either mono-culture or co-culture with neurons or astrocytes, are a powerful tool for studying mechanisms underlying microglial inflammatory responses and cell type-specific interactions in the central nervous system (CNS). This protocol provides the details of how to prepare high purity primary microglia from newborn mouse pups. The overall steps include brain cell dissociation, mixed glial cell culture, and microglia isolation.
[Background] In recent years, neuroinflammation has become a hotspot area in neuroscience studies. Inflammatory responses, such as glial activation and cytokine upregulation, were observed in brains of patients with various neurological diseases (Fan et al., 2015; Koshimori et al., 2015; Garden and Campbell, 2016). Neuroinflammation is considered not only a consequence of pathological changes in the brain but also a contributor to disease progression (Schwartz et al., 2013). In addition, the physiological functions of inflammatory pathways, the importance of which were previously underestimated, are being revealed as surprisingly versatile. For instance, activation of the complement signaling pathway is commonly observed in the central nervous system (CNS) in neurological diseases and is suspected to be involved in disease pathophysiology (Michailidou et al., 2015; Loeffler et al., 2008). Now we know that it also plays essential function in the developmental regulation of synaptic refinement (Stevens et al., 2007). Along with the increasing attention on inflammation, interest in microglial function during development, neuroprotection, and pathogenesis continues growing. Microglia are resident innate immune cells of myeloid lineage located in the brain and are critical components of the immune system in the CNS. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. For instance, TREM2 mutations, which affects only microglia, are a genetic risk factor for Alzheimer’s disease (Yuan et al., 2016; Wang et al., 2015). At the same time, developmental roles of microglia are being revealed. For example, synaptic maturation during early development requires microglia and this regulation may underline the pathogenesis of developmental diseases such as autism (Edmonson et al., 2016; Stephan et al., 2012). Tools for studying microglia include in vivo models (e.g., microglia-deficient PU.1 knockout mice [McKercher et al., 1996]) and in vitro systems such as immortalized microglial cell lines and primary microglial culture. While in vivo tools are powerful for demonstrating systematic microglial function, in vitro tools are ideal for mechanistic characterization due to the easy manipulation of experimental factors. Compared to immortalized microglial cell lines, primary microglia better mimic in vivo microglial properties (Stansley et al., 2012). The altered gene expression upon stimulation may be better presented in primary microglia than in microglial cell lines (Stansley et al., 2012; Henn et al., 2009). Here we described a protocol for establishing high purity primary microglial culture derived from neonatal mice and the method has yielded robust results in our work (Lian et al., 2016). Dissociated cells are collected through enzymatic digestion of collected brains and seeded to grow mixed glial culture. Microglia growing on top of a confluent astrocyte layer are purified through mechanical tapping of mixed glial culture.
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