Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia
Minimizing the Ex Vivo Confounds of Cell-Isolation Techniques on Transcriptomic and Translatomic Profiles of Purified Microglia
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DOI：
10.1523/eneuro.0348-21.2022
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发表时间：
2022-03-01
期刊：
影响因子：
3.4
通讯作者：
Freeman, Willard M.
中科院分区：
文献类型：
--
作者：
Ocanas, Sarah R.;Pham, Kevin D.;Freeman, Willard M.
现代分子和生化神经科学研究需要对来自脑组织样本的特定细胞群体进行分析，以消除细胞类型特定事件的歧义。这在对大脑中少数神经胶质细胞群体的分析中尤其如此，例如在整个组织分析中可能被遮盖的小胶质细胞。小胶质细胞在发育、衰老和神经退行性变中具有重要作用，是当前神经科学研究的热点。长期以来，使用活体模型的神经胶质生物学家一直关注的一个问题是，从中枢神经系统组织中分离细胞是否会在小胶质细胞中引入体外人工制品，小胶质细胞对环境变化做出快速反应。通过磁激活细胞分选(MACS)以及基于细胞仪和基于墨盒的荧光激活细胞分选(FACS)方法纯化小鼠小胶质细胞，以比较和对比其性能。CX3CR1-NuTRAP小鼠模型被用来提供内源性荧光小胶质细胞标记物和小胶质细胞特异性翻译组作为基线比较，缺少细胞分离伪影。所有分选方法在小胶质细胞纯度方面都进行了相似的操作，主要区别在于细胞产量和分离时间。体外激活信号主要发生在最初的组织分离和细胞准备过程中，而不是细胞分选过程中。细胞准备诱导的激活表型可以通过加入转录和翻译抑制物或完全在低温下进行非酶解离来最小化。这些数据表明，根据实验需要，可以使用各种小胶质细胞分离方法，并且抑制剂鸡尾酒在减少细胞准备伪影方面是有效的。
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.