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Author:
Date:
2015-01-20
[Abstract] Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators.
In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating ...
[摘要] 单细胞分析已成为免疫学中重要的一种方法。荧光流式细胞仪一直是主要的参与者。然而，由于诸如自身荧光和不同荧光团之间的发射溢出的问题，正在开发替代技术。近年来，已经出现了大量细胞计数，其中用金属离子标记的抗体通过ICP-MS检测。为了看到电池，必须存在质量窗口中的金属;没有与前向或侧向散射的类似参数。选择的当前质量窗口大约是AW 103-196，其包括用于大多数抗体标记的镧系元素，以及用于DNA嵌入剂的铱和铑。在本方案中，我们使用用MAXPAR金属标记的抗体混合物螯合聚合物对先前冷冻保存的表面染色活的PBMC。许多这些标记取自标准荧光表型组（Maecker等人，2012）。不使用细胞内抗体。我们使用CyTOF TM （通过飞行时间细胞计数）质谱仪获取ICP-MS数据。使用FlowJo软件的双计数信号数据的后续分析允许基于每个质量通道中的双计数信号来分析细胞类型。确定每种细胞类型的百分比，并报告为父细胞类型的百分比。
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