Mazzolini Julie, Chia Kelda, Sieger Dirk
Centre for Discovery Brain Sciences, University of Edinburgh.
Centre for Discovery Brain Sciences, University of Edinburgh;
J Vis Exp. 2018 Apr 27(134):57431. doi: 10.3791/57431.
To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody. Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 °C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 °C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) > 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.
为了深入了解不同中枢神经系统（CNS）细胞在发育过程中或脑部疾病的发生与发展过程中的作用，在不改变其基因表达谱的情况下分离这些细胞至关重要。斑马鱼模型提供了大量转基因鱼系，其中特定细胞类型被标记；例如，NBT:DsRed系中的神经元或mpeg1:eGFP系中的巨噬细胞/小胶质细胞。此外，已开发出抗体来标记特定细胞，如用4C4抗体标记小胶质细胞。在此，我们描述了从斑马鱼幼体脑中分离神经元、巨噬细胞和小胶质细胞的方法。该方案的核心是避免在37°C进行酶促组织消化，因为这可能会改变细胞特征。相反，使用了在4°C进行组织匀浆的机械系统。该方案包括将脑匀浆成细胞悬液、对其进行免疫染色以及通过荧光激活细胞分选（FACS）分离神经元、巨噬细胞和小胶质细胞。之后，我们从这些细胞中提取RNA并评估其质量/数量。我们成功获得了高质量的RNA（RNA完整性数值（RIN）>7），用于对巨噬细胞/小胶质细胞和神经元进行定量聚合酶链反应（qPCR），以及对小胶质细胞进行转录组分析。这种方法能够更好地表征这些细胞，并更清楚地了解它们在发育和疾病中的作用。
