Abstract
Modern molecular and biochemical neuroscience studies require analysis of specific cellular populations derived from brain tissue samples to disambiguate cell type-specific events. This is particularly true in the analysis of minority glial populations in the brain, such as microglia, which may be obscured in whole tissue analyses. Microglia have central functions in development, aging, and neurodegeneration and are a current focus of neuroscience research. A long-standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment. Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer-based and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance. The Cx3cr1-NuTRAP mouse model was used to provide an endogenous fluorescent microglial marker and a microglial-specific translatome profile as a baseline comparison lacking cell isolation artifacts. All sorting methods performed similarly for microglial purity with main differences being in cell yield and time of isolation. Ex vivo activation signatures occurred principally during the initial tissue dissociation and cell preparation and not the cell sorting. The cell preparation-induced activational phenotype could be minimized by inclusion of transcriptional and translational inhibitors or non-enzymatic dissociation conducted entirely at low temperatures. These data demonstrate that a variety of microglial isolation approaches can be used, depending on experimental needs, and that inhibitor cocktails are effective at reducing cell preparation artifacts.
Highlights
Microglia, the brain’s resident macrophages, have come to the forefront of neuroimmunology research (Prinz et al, 2019)
A long66 standing concern for glial biologists using in vivo models is whether cell isolation from CNS tissue could introduce ex vivo artifacts in microglia, which respond quickly to changes in the environment
Mouse microglia were purified by magnetic-activated cell sorting (MACS), as well as cytometer- and cartridge-based fluorescence-activated cell sorting (FACS) approaches to compare and contrast performance
Summary
The brain’s resident macrophages, have come to the forefront of neuroimmunology research (Prinz et al, 2019). They serve as surveyors of the central nervous system and exhibit behavior derived from their embryonic precursors, myeloid cells (Cuadros and Navascues, 1998; Rock et al, 2004), with roles in neurodevelopment, sex differences, as well as in health and neurodegenerative diseases (Butovsky and Weiner, 2018; Han et al, 2021; Salter and Stevens, 2017). Microglial cells transition to an activated phenotype, classically defined by morphological transformation from ramified to amoeboid, release of pro-inflammatory cytokines, and a shift in global gene expression (Avignone et al, 2015; Rock et al, 2004; Sierra et al, 2013). To delineate the full heterogeneity of microglial population with aging and disease using single cell techniques, it is necessary to minimize activational effects of cell preparation and isolation
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