Characteristics of primary rat microglia isolated from mixed cultures using two different methods.
Characteristics of primary rat microglia isolated from mixed cultures using two different methods.
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DOI：
10.1186/s12974-017-0877-7
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发表时间：
2017-05-08
影响因子：
9.3
通讯作者：
Xing C
中科院分区：
文献类型：
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作者：
Lin L;Desai R;Wang X;Lo EH;Xing C
小胶质细胞培养是研究几乎所有中枢神经系统疾病炎症机制的一个至关重要的模型系统。从混合胶质细胞培养中分离原代小胶质细胞最常用的两种方法是轻度胰蛋白酶化和摇法。在本研究中，我们对这两种方法获得的小胶质细胞进行了表征和比较。从1 - 2日龄的新生Sprague-Dawley大鼠大脑皮层制备大鼠小胶质细胞原代培养物。在体外约14天达到融合后，通过轻度胰蛋白酶化或摇晃从混合胶质细胞培养物中分离小胶质细胞。流式细胞术检测小胶质细胞纯度。采用实时荧光定量PCR检测mRNA表达。采用ELISA试剂盒检测细胞培养上清中TNFα、IL-1β、IL-10和IGF-1的含量。使用荧光素标记的大肠杆菌K-12生物颗粒评估吞噬功能。温和胰蛋白酶化比摇匀产生更高的产量和纯度。经轻度胰蛋白酶化分离的小胶质细胞处于静止状态，形态呈分枝状。通过摇动分离的小胶质细胞表现出更异质的形态，包括圆形的细胞，暗示着激活。与摇晃相比，胰蛋白酶化分离的小胶质细胞也具有较低的基线表型标记（iNOS, CD86， CD206和精氨酸酶1）和较低的细胞因子（TNFα, IL-1β， IL-10和IGF-1）水平，以及降低的吞噬能力。两种方法产生的小胶质细胞对各种刺激，如IL-4、脂多糖（LPS）或干扰素-γ (IFNγ)有反应。虽然受刺激的基因表达和细胞因子释放模式大致相似，但在绝对反应方面也存在显著差异。LPS处理显著提高了轻度胰蛋白酶化分离的小胶质细胞中TNFα和IL-10的水平。在轻度胰蛋白酶化获得的小胶质细胞中，IFNγ诱导的TNFα反应较低。我们的研究结果表明，用震动法分离小胶质细胞即使在基线时也会引起轻微的激活，这可能会影响后续实验中的刺激反应。在选择原代小胶质细胞培养的分离方案时，应谨慎和注意。
Microglial cultures comprise a critically important model system for investigating inflammatory mechanisms in almost all CNS disorders. Mild trypsinization and shaking are the two most commonly used methods to isolate primary microglia from mixed glial cultures. In this study, we characterized and compared microglia obtained using these two methods. Primary rat microglia cultures were prepared from cerebral cortices of 1–2-day-old neonatal Sprague-Dawley rats. After achieving confluency at about 14 days in vitro, microglia were isolated from mixed glial cultures via either mild trypsinization or shaking. The purity of microglia was estimated by flow cytometry. Quantitative real-time PCR was used to measure mRNA expression. TNFα, IL-1β, IL-10, and IGF-1 in cell culture supernatant were measured using ELISA kits. Phagocytic function was assessed using fluorescein-labeled Escherichia coli K-12 BioParticles. Mild trypsinization generated a higher yield and purity than shaking. Microglia isolated by mild trypsinization appeared to be in a quiescent state with ramified morphology. Microglia isolated by shaking showed a more heterogenous morphology, including cells with rounded shapes suggestive of activation. Compared with shaking, microglia isolated by trypsinization also had lower baseline phenotype markers (iNOS, CD86, CD206, and arginase 1) and lower levels of cytokines (TNFα, IL-1β, IL-10, and IGF-1) as well as reduced phagocytic capability. Both methods yielded microglia that were responsive to various stimuli such as IL-4, lipopolysaccharide (LPS), or interferon-γ (IFNγ). Although stimulated patterns of gene expression and cytokine release were generally similar, there were also significant differences in terms of absolute response. LPS treatment induced significantly higher levels of TNFα and IL-10 in microglia isolated by mild trypsinization versus shaking. IFNγ induced a lower response in TNFα in microglia obtained by mild trypsinization versus shaking. Our results suggest that isolating microglia with the shaking method may induce slight activation even at baseline, and this may affect stimulus responses in subsequent experiments. Caution and attention should be warranted when choosing isolation protocols for primary microglia cultures.