Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis
Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis
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DOI：
10.1371/journal.pone.0068519
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发表时间：
2013-06-24
期刊：
影响因子：
3.7
通讯作者：
Pruszak, Jan
中科院分区：
文献类型：
--
作者：
Turac, Gizem;Hindley, Christopher J.;Pruszak, Jan
表面分子谱在生理学和病理学中经历动态变化，用作细胞状态和表型的标记，并且可以用于细胞选择策略和诊断。明确定义的细胞亚群的分离是干细胞生物学中体内和体外应用所需要的。在这份技术报告中，我们提出了一种通过流式细胞术检测细胞内抗原来确定混合细胞群中感兴趣的子集的方法。我们已经开发了一个完全验证的协议，使共同检测的分化簇（CD）表面抗原的固定，透化的神经细胞群定义的细胞内染色。在神经母细胞瘤细胞系（SH-SY 5 Y，BE（2）-M17）上确定表面标志物候选物与细胞内靶群体标志物（巢蛋白、MAP 2、双皮质素、TUJ 1）的共表达程度，产生组合的CD 49 f-/CD 200（高）表面标志物组。其在荧光激活细胞分选（FACS）中的应用从源自人诱导多能干细胞的分化细胞悬浮液产生富集的神经元培养物。我们的数据强调了使用所描述的共标记方案和共表达分析用于哺乳动物神经生物学中的定量测定和用于筛选方法以鉴定干细胞生物学中急需的表面标志物的可行性。
Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f-/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology.