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Author:
Date:
2015-01-05
[Abstract] In this protocol, we use a CyTOFTM mass cytometry to collect single-cell data on a large number of cytokines/chemokines as well as cell-surface proteins that characterize T cells and other immune cells. The current selected mass window in AW 103-203 includes the lanthanides used for most antibody labeling, along with iridium and rhodium for DNA intercalators. The output data are in the format as .txt and .fcs files, which is compatible with many analysis programs. This protocol could be adapted to include tetramers into the staining panel, but we have not optimized for that purpose.
The principal steps of intracellular cytokine staining are as follows: First, cells are activated for a few hours using either a specific peptide or a non-specific activation cocktail. ...
[摘要] 在这个协议中，我们使用CyTOF TM 质谱仪收集大量细胞因子/趋化因子以及表征T细胞和其他免疫细胞的细胞表面蛋白的单细胞数据。 AW 103-203中当前选择的质量窗口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。输出数据的格式为.txt和.fcs文件，与许多分析程序兼容。该方案可以适于将四聚体包括在染色板中，但是我们没有为此目的进行优化。细胞内细胞因子染色的主要步骤如下：首先，细胞被活化几小时，使用特异性肽或非特异性活化混合物。加入蛋白质转运抑制剂（例如布雷菲德菌素A）以将细胞因子保留在细胞内。接下来，加入EDTA以从活化容器中除去粘附细胞。洗涤后，将细胞表面标记物的抗体加入细胞中。然后将细胞固定在多聚甲醛中并透化。我们使用温和的洗涤剂皂苷作为渗透缓冲液，因为与苛刻的洗涤剂或甲醇相比，它对表面和细胞内表位的破坏性更小。透化后，将金属缀合的抗细胞因子抗体加入细胞悬浮液中。然后将染色的细胞依次导入质谱仪进行信号强度分析
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