Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia.

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.