Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord
Isolation, culture, and downstream characterization of primary microglia and astrocytes from adult rodent brain and spinal cord
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DOI：
10.1016/j.jneumeth.2020.108742
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发表时间：
2020-07-01
影响因子：
3
通讯作者：
Burton, Michael D.
中科院分区：
文献类型：
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作者：
Agalave, Nilesh M.;Lane, Brandon T.;Burton, Michael D.
背景：神经免疫学家渴望了解中枢神经系统中神经元、小胶质细胞和星形胶质细胞之间的相互作用。为了研究这些细胞，研究人员使用永生化细胞系或从动物组织中获得的原代细胞。原代细胞反映在体内的特性和功能相比，永生化的细胞，但是，他们是具有挑战性的收购和maintain.New方法：建立协议，收获初级胶质细胞使用新生啮齿动物，在这里，我们提供了一种方法，同时分离小胶质细胞和星形胶质细胞从成年啮齿动物的大脑和/或脊髓。我们利用了一个不连续的CD 11密度梯度，使这些细胞群体的容易区分，没有酶消化或复杂的分选techniques.Results：我们发现从CD 11接口分离的细胞70%- 50%之间的小胶质细胞，因为它们表达电离钙结合接头分子1（Iba 1）在免疫细胞化学和CD 11b（hi）和CD 45（lo）使用流式细胞术。从50%-30%界面分离的细胞是星形胶质细胞，因为它们在免疫细胞化学中表达胶质细胞酸性蛋白（GFAP），并使用流式细胞术表达谷氨酸天冬氨酸转运蛋白（GLAST）1。培养的小胶质细胞和星形胶质细胞在脂多糖（LPS）处理后显示出IL-6产生的功能性增加。与现有方法的比较：我们的方法允许在一种方案中快速分离小胶质细胞和星形胶质细胞，资源相对较少，保留细胞表型，并且无需磁性或抗体分选即可获得高细胞数量。结论：在这里，我们展示了一种新颖的单一方案，从脑和脊髓组织中分离小胶质细胞和星形胶质细胞，允许从不同年龄的动物细胞中培养和其他下游应用，这将有助于研究人员从同一啮齿动物的大脑或脊髓中研究这两种主要的神经胶质细胞类型。
Background: Neuroimmunologists aspire to understand the interactions between neurons, microglia, and astrocytes in the CNS. To study these cells, researchers work with either immortalized cell lines or primary cells acquired from animal tissue. Primary cells reflect in vivo characteristics and functionality compared to immortalized cells; however, they are challenging to acquire and maintain.New Method: Established protocols to harvest primary glia use neonatal rodents, here we provide a method for simultaneously isolating microglia and astrocytes from brain and/or spinal cord from adult rodents. We utilized a discontinuous percoll density gradient enabling easy discrimination of these cell populations without enzymatic digestion or complex sorting techniques.Results: We found cells isolated from the percoll interface between 70%- 50% were microglia, as they express ionizing calcium-binding adaptor molecule 1 (Iba1) in immunocytochemistry and CD11b(hi) and CD45(lo) using flow cytometry. Isolated cells from the 50 %-30 % interface were astrocytes as they express glial fibrillary acidic protein (GFAP) in immunocytochemistry and Glutamate aspartate transporter (GLAST) 1 using flow cytometry. Cultured microglia and astrocytes showed a functional increase in IL-6 production after treatment of lipopolysaccharide (LPS).Comparison with Existing Methods: Our method allows for rapid isolation of both microglia and astrocytes in one protocol with relatively few resources, preserves cellular phenotype, and yields high cell numbers without magnetic or antibody sorting.Conclusion: Here we show a novel, single protocol to isolate microglia and astrocytes from brain and spinal cord tissue, allowing for culturing and other downstream applications from the cells of animals of various ages, which will be useful for researchers investigating these two major glial cell types from the brain or spinal cord of the same rodent.