Establishment and Characterization of Primary Adult Microglial Culture in Mice
Establishment and Characterization of Primary Adult Microglial Culture in Mice
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小鼠原代成年小胶质细胞培养物的建立和表征
DOI：
10.1007/978-3-7091-1434-6_8
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Shioda S
中科院分区：
文献类型：
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作者：
Ohtaki H;Tsumuraya T;Song D;Sato A;Ohara K;Miyamoto K;Nakano H;Kiriyama K;Dohi K;Hiraizumi H;Matsunaga M;Shioda S
小胶质细胞约占中枢神经系统（CNS）细胞的12- 15%。小胶质细胞在细胞因子、趋化因子和生长因子等病理刺激下发生极化，在中枢神经系统的损伤和修复中发挥重要作用。在这里，我们建立了原代小胶质细胞的培养分离的成年C57/BL 6小鼠的大脑使用Percoll密度梯度。培养细胞并用抗CD 11b、胶质细胞酸性蛋白、髓鞘碱性蛋白和NeuN的抗体染色，以分别确定小胶质细胞、星形胶质细胞、少突胶质细胞和神经元细胞。此外，将细胞暴露于干扰素-γ（IFNγ）加白细胞介素-1 β（IL-1β）或IL-4 24 h，以用诱导型一氧化氮合酶（iNOS）、Ym 1和Iba-1免疫印迹证明活化表型。至少95%的培养细胞为CD 11b阳性，星形胶质细胞、神经元和少突胶质细胞标记物为阴性。IFNγ + IL-1β处理导致经典活化，其表现为iNOS的增加。细胞还显示出交替激活，当用IL-4处理时，其增加Ym 1。本研究表明，小胶质细胞分离，如这里所描述的是一个有用的工具，阐明成人小胶质细胞的功能。
Microglial cells account for approximately 12–15 % of the cells in the central nervous system (CNS). Microglial cells are polarized by pathological stimuli such as cytokines, chemokines, and growth factors, and play important roles in the deterioration and repair of the CNS. Here, we established cultures of primary microglial cells isolated from the brains of adult C57/BL6 mice using Percoll density gradients. The cells were cultured and stained with antibodies against CD11b, glial fibrillary acidic protein, myelin basic protein and NeuN to determine microglial, astroglial, oligodendroglial, and neuronal cells respectively. Moreover, the cells were exposed to interferon-γ (IFNγ) plus interleukin-1β (IL-1β) or IL-4 for 24 h to demonstrate the activating phenotypes with inducible nitric oxide synthase (iNOS), Ym1, and Iba-1 immunoblotting. At least 95 % of the cultured cells were CD11b-positive and -negative for astroglial, neuronal, and oligodendrocyte markers. IFNγ plus IL-1β treatment resulted in classical activation, which was represented by an increase in iNOS. The cells also displayed alternative activation, which increased Ym1 when treated with IL-4. The present study indicates that the microglial cells isolated as described here are a useful tool for elucidating adult microglial function.