High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.
High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.
复制标题
用于直接评估哺乳动物细胞中 GM1 神经节苷脂水平的高通量成像方法。
DOI：
10.1016/j.dib.2016.01.027
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发表时间：
2016
期刊：
影响因子：
1.2
通讯作者：
Cramer,CaroleL
中科院分区：
文献类型：
--
作者：
Acosta,Walter;Martin,Reid;Radin,DavidN;Cramer,CaroleL
神经节苷脂沉积症是一种遗传性常染色体隐性遗传性疾病，由编码酸性β-半乳糖苷酶(β-GAL)的GLB1基因突变引起。这种溶酶体酶缺乏活性导致神经节苷脂(GM1)在细胞内积累。我们已经开发了一种高含量成像方法来评估成纤维细胞中的GM1水平，该方法可用于评估经处理的GLB1−/−细胞的底物减少[1]。这种分析允许在多井系统中进行荧光定量，从而产生无偏倚和统计上有意义的数据。用荧光标记的霍乱毒素B亚单位(CTXB)与神经节苷脂GM1结合，检测成纤维细胞中GM1的水平。这种灵敏、快速和廉价的方法便于在体外药物筛选中以一种允许使用低工作量进行大量重复的格式进行筛选。
GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the geneGLB1, which encodes acid β-galactosidase (β-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1−/−cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detectin situGM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitatesin vitrodrug screening in a format that allows a high number of replicates using low working volumes.