| Phenotyping of Live Human PBMC using CyTOFTM Mass Cytometry
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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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| Intracellular Cytokine Staining on PBMCs Using CyTOFTM Mass Cytometry
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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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| In vitro Regulatory T cells Differentiation From Naïve T Cells
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Author:
Date:
2014-03-20
[Abstract] In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant immune responses to foreign antigens. Remarkably, regulatory T cells have been implicated in tumor cell evasion of immune responses (Curiel et al., 2004; Zou, 2006) by suppressing T cell mediated antitumor immunity. The study of the signals that promote the differentiation of this suppressive population in the tumor microenvironment has become a central issue. Here we described a detailed method to in vitro differentiate Tregs using ...
[摘要] 在过去的几年中，表达CD4，CD25和转录因子FoxP3的调节性T细胞（Treg）的一个子集已经作为T细胞耐受性和体内平衡的关键调节剂获得了相当大的关注（Sakaguchi，2004）。 这种T细胞群特别参与维持免疫自身耐受性和控制对外来抗原的异常免疫应答。 值得注意的是，调节性T细胞通过抑制T细胞介导的抗肿瘤免疫参与了肿瘤细胞逃避免疫应答（Curiel等人，2004; Zou，2006）。 促进这种抑制群体在肿瘤微环境中的分化的信号的研究已经成为一个中心问题。 在这里，我们描述了使用来自小鼠天然T细胞的肿瘤细胞条件培养基来体外鉴别Treg的详细方法，并且基于它们的特异性标记物来鉴定它们（Dalotto-Moreno等人， >，2013）。
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