Magnetic cell sorting: A fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue
Magnetic cell sorting: A fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue
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DOI：
10.1016/j.jneumeth.2008.08.016
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发表时间：
2008-10-30
影响因子：
3
通讯作者：
Sarma, Jayasri Das
中科院分区：
文献类型：
--
作者：
Marek, Ryan;Caruso, Michael;Sarma, Jayasri Das
星形胶质细胞和小胶质细胞功能的病理改变在中枢神经系统（CNS）疾病中起着关键作用。中枢神经系统的复杂性使得很难在体内确定单个神经胶质细胞的功能。深入了解单个神经胶质细胞功能的作用在于它们的成功分离和纯化，以保持表型并真实地模拟体内条件。为了促进这样的实验，我们设计了一个单一的步骤神经胶质细胞分离程序的基础上抗原抗体介导的磁性细胞分选，其中个别的神经胶质细胞群体富集阳性选择或从相同的混合神经胶质细胞培养耗尽。我们通过抗体介导的细胞溶解从混合胶质细胞培养物中去除少突胶质细胞污染，并将剩余的细胞应用于磁场中的CD 11b微珠。从CD 11b柱中，我们通过阳性选择分离小胶质细胞，通过消耗小胶质细胞分离星形胶质细胞。通过阳性选择分离的小胶质细胞>99%纯且不含星形胶质细胞，而通过阴性选择收集的星形胶质细胞为95-97%纯且完全不含小胶质细胞。这种改进的技术是简单，快速，通用，方便和可靠的分离单个胶质细胞群体从单一的混合胶质细胞培养物的基础上细胞特异性抗原抗体相互作用。随后，这些培养物可用于在形态学和分子水平上研究单个胶质细胞在正常和病理条件下的功能。(C)2008 Elsevier B. V.保留所有权利。
Pathologically altered functions of astrocytes and microglia play a pivotal role in diseases of the central nervous system (CNS). The complexity of the CNS makes it difficult to determine the function of individual glial cells in vivo. Insight into the role of individual glial cell function lies in their successful isolation and purification to maintain phenotype and realistically mimic in vivo conditions. To facilitate such experiments we have designed a single-step glial cell isolation procedure based on antigen antibody-mediated magnetic cell sorting whereby individual glial cell populations are enriched by positive selection or by depletion from the same mixed glial culture. We removed oligodendroglial contamination from mixed glial culture by antibody-mediated cytolysis, and applied the remaining cells to CD11b MicroBeads in a magnetic field. From the CD11b column we isolated microglia by positive selection and astrocytes by depleting microglia. Microglia isolated by positive selection were >99% pure and free from astrocytes, while astrocytes collected by negative selection were 95-97% pure and completely free from microglia. This modified technique is simple, fast, versatile, convenient and reliable for the isolation of individual glial cell populations from single mixed glial cultures based on cell-specific antigen-anti body interaction. Subsequently, these cultures can be applied to study the function of individual glial cells at the morphological and molecular level during normal and pathological condition. (C) 2008 Elsevier B.V. All rights reserved.